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Sample records for bact lactis aerogenes

  1. Debunking BACT

    SciTech Connect

    Finto, K.; Harrison, C.; Lomax, S.

    2006-11-15

    The US Clean Air Act's provisions for the Prevention of Significant Deterioration (PSD) of air quality require a new major stationary source to obtain a preconstruction permit that specifies the Best Available Control Technology (BACT) for each regulated pollutant that may be emitted in amounts greater than major source thresholds. The PSD regulations also impose BACT requirements on modifications to existing major sources that result in significant net emissions increases. Rather than a specific technology, BACT is an achievable emissions limitation (or work practice) determined by the permitting authority on a case-by- case basis, taking into account available technologies and energy, environmental, and economic impacts. BACT determinations are generally made by a state environmental agency after an opportunity for public comment. Increasingly, advocacy groups are challenging BACT decisions in administrative and judicial proceedings. As a result, the permitting process has been substantially delayed, even for facilities that have agreed to install state-of-the-art emissions control technology. This article outlines the key statutory and regulatory elements of BACT, how to analyze alternative technologies and emissions limitations, and prepare an application for an appropriate and final BACT determination. It is based largely on the regulatory definition of BACT and recent Environmental Appeals Board (EAB) decisions. Case-by-case BACT analyses do not necessarily yield a single, objectively correct BACT determination. The permitting agency must exercise a high degree of technical judgment in any BACT analysis, particularly for coal-fired plants, which use a wide variety of coals, combustion techniques, and other site-specific factors. 12 refs.

  2. Bioengineering of the Enterobacter aerogenes strain for biohydrogen production.

    PubMed

    Zhang, Chong; Lv, Feng-Xiang; Xing, Xin-Hui

    2011-09-01

    Enterobacter aerogenes is one of the most widely-studied model strains for fermentative hydrogen production. To improve the hydrogen yield of E. aerogenes, the bioengineering on a biomolecular level and metabolic network level is of importance. In this review, the fermentative technology of E. aerogenes for hydrogen production will be first briefly summarized. And then the bioengineering of E. aerogenes for the improvement of hydrogen yield will be thoroughly reviewed, including the anaerobic metabolic networks for hydrogen evolution in E. aerogenes, metabolic engineering for improving hydrogen production in E. aerogenes and mixed culture of E. aerogenes with other hydrogen-producing bacteria to enhance the overall yield in anaerobic cultivation. Finally, a perspective on E. aerogenes as a hydrogen producer including systems bioengineering approach for improving the hydrogen yield and application of the engineered E. aerogenes in mixed culture will be presented.

  3. Dechlorination of DDT by Aerobacter aerogenes

    USGS Publications Warehouse

    1966-01-01

    Dechlorination of DDT to DDD in higher animals requires the presence of molecular oxygen, but in microorganisms the presence of oxygen hinders dechlorination. In cell-free preparations of Aerobacter aerogenes, the use of selected metabolic inhibitors indicated that reduced Fe(II) cytochrome oxidase was responsible for DDT dechlorination. This finding may possibly explain. the persistence of DDT residues in soils and sediments.

  4. The aerogen-π bonds involving π systems

    NASA Astrophysics Data System (ADS)

    Gao, Meng; Cheng, Jianbo; Li, Wenzuo; Xiao, Bo; Li, Qingzhong

    2016-05-01

    Ab initio calculations have been performed to study the complexes of XeOF2 and a series of π systems including ethyne, ethene, benzene, pyrrole, furan, and thiophene. More than two structures were obtained for each complex with an aerogen-π bond. The configuration of complex has a significant effect on its stability. The strength of aerogen-π interaction is comparable with that of lone pair-aerogen interaction and conventional hydrogen bonds. A breakdown of the aerogen-π interaction attributes its stability to approximately equal parts electrostatic and polarization energies, with a relatively smaller contribution from dispersion energy.

  5. Complete genome sequence of Enterobacter aerogenes KCTC 2190.

    PubMed

    Shin, Sang Heum; Kim, Sewhan; Kim, Jae Young; Lee, Soojin; Um, Youngsoon; Oh, Min-Kyu; Kim, Young-Rok; Lee, Jinwon; Yang, Kap-Seok

    2012-05-01

    This is the first complete genome sequence of the Enterobacter aerogenes species. Here we present the genome sequence of E. aerogenes KCTC 2190, which contains 5,280,350 bp with a G + C content of 54.8 mol%, 4,912 protein-coding genes, and 109 structural RNAs.

  6. Complete Genome Sequence of Enterobacter aerogenes KCTC 2190

    PubMed Central

    Shin, Sang Heum; Kim, Sewhan; Kim, Jae Young; Lee, Soojin; Um, Youngsoon; Oh, Min-Kyu; Kim, Young-Rok; Lee, Jinwon

    2012-01-01

    This is the first complete genome sequence of the Enterobacter aerogenes species. Here we present the genome sequence of E. aerogenes KCTC 2190, which contains 5,280,350 bp with a G + C content of 54.8 mol%, 4,912 protein-coding genes, and 109 structural RNAs. PMID:22493190

  7. Transmission of Enterobacter aerogenes septicemia in healthcare workers.

    PubMed

    Jha, Piyush; Kim, Choon-Mee; Kim, Dong-Min; Chung, Jong-Hoon; Yoon, Na-Ra; Jha, Babita; Kim, Seok Won; Jang, Sook Jin; Ahn, Young-Joon; Chung, Jae Keun; Jeon, Doo Young

    2016-01-01

    Enterobacter aerogenes is recognized as an important bacterial pathogen in hospital-acquired infections. This report describes two unusual cases of septicemia caused by E. aerogenes in immunocompetent healthcare workers. E. aerogenes was isolated from blood cultures of the two patients experiencing septicemia. The clinical isolates were initially identified as E. aerogenes using a VITEK II automated system and 16S rRNA sequence analysis, and; both isolates involved in the outbreak shared a common pulse-field gel electrophoresis pattern. The similarities between the two cases included the simultaneous development of gastroenteritis symptoms, severe sepsis and thrombocytopenia after taking intravenous injections of ketorolac tromethamine. A common source of normal saline, a 100 mL bottle, was used for diluting the analgesic in both cases. In addition to the general population, healthcare workers, especially those who are also intravenous drug abusers, should be considered subjects that could cause a transmission of Enterobacter infection.

  8. Transmission of Enterobacter aerogenes septicemia in healthcare workers.

    PubMed

    Jha, Piyush; Kim, Choon-Mee; Kim, Dong-Min; Chung, Jong-Hoon; Yoon, Na-Ra; Jha, Babita; Kim, Seok Won; Jang, Sook Jin; Ahn, Young-Joon; Chung, Jae Keun; Jeon, Doo Young

    2016-01-01

    Enterobacter aerogenes is recognized as an important bacterial pathogen in hospital-acquired infections. This report describes two unusual cases of septicemia caused by E. aerogenes in immunocompetent healthcare workers. E. aerogenes was isolated from blood cultures of the two patients experiencing septicemia. The clinical isolates were initially identified as E. aerogenes using a VITEK II automated system and 16S rRNA sequence analysis, and; both isolates involved in the outbreak shared a common pulse-field gel electrophoresis pattern. The similarities between the two cases included the simultaneous development of gastroenteritis symptoms, severe sepsis and thrombocytopenia after taking intravenous injections of ketorolac tromethamine. A common source of normal saline, a 100 mL bottle, was used for diluting the analgesic in both cases. In addition to the general population, healthcare workers, especially those who are also intravenous drug abusers, should be considered subjects that could cause a transmission of Enterobacter infection. PMID:27610316

  9. Partial hydrolysis of dieldrin by Aerobacter aerogenes

    USGS Publications Warehouse

    Wedemeyer, G.

    1968-01-01

    Although dieldrin (1,2,3 ,4,10,10-hexachloro- 6,7-epoxy-1 ,4 ,4a ,5 ,6 ,7 ,8, 8a-octahydro-1 ,4-endo, exo-5, 8-dimethanonaphthalene) metabolism by mammals (F. Korte and H. Arent, Life Sci. 4:2017, 1965) and insects (D. F. Heath and M. Vanderkar, Brit. J. Ind. Med. 21:269, 1964) has been reported, little is known about the degradation of this important pesticide by microorganisms. Korte et al. (Ann. Chem. Liebigs 656:135, 1962) and Chacko et al. (Science 154: 893, 1966) reported that a number of ubiquitous microorganisms were incapable of degrading dieldrin; however, more recently Matsumura and Boush (Science 156:959, 1967) isolated several species of Pseudomonas and Bacillus which could degrade dieldrin, from a number of soil samples having similar activity. They did not specifically attempt to identify the dieldrin metabolites formed, but they did suggest, on the basis of an identical RF value with an authentic control that 6,7-trans-dihydroxydihydroaldrin (aldrin diol) might be a major product. Work carried out concurrently in this laboratory has shown that another ubiquitous bacterium, Aerobacter aerogenes, converts dieldrin in vitro to a compound chromatographically

  10. Utilization of arginine by Klebsiella aerogenes.

    PubMed

    Friedrich, B; Magasanik, B

    1978-02-01

    Klebsiella aerogenes utilized arginine as the sole source of carbon or nitrogen for growth. Arginine was degraded to 2-ketoglutarate and not to succinate, since a citrate synthaseless mutant grows on arginine as the only nitrogen source. When glucose was the energy source, all four nitrogen atoms of arginine were utilized. Three of them apparently did not pass through ammonia but were transferred by transamination, since a mutant unable to produce glutamate by glutamate synthase or glutamate dehydrogenase utilized three of four nitrogen atoms of arginine. Urea was not involved as intermediate, since a unreaseless mutant did not accumulate urea and grew on arginine as efficiently as the wild-type strain. Ornithine appeared to be an intermediate, because cells grown either on glucose and arginine or arginine alone could convert arginine in the presence of hydroxylamine to ornithine. This indicates that an amidinotransferase is the initiating enzyme of arginine breakdown. In addition, the cells contained a transaminase specific for ornithine. In contrast to the hydroxylamine-dependent reaction, this activity could be demonstrated in extracts. The arginine-utilizing system (aut) is apparently controlled like the enzymes responsible for the degradation of histidine (hut) through induction, catabolite repression, and activation by glutamine synthetase.

  11. Draft Genome Sequence of Lactococcus lactis subsp. lactis Strain YF11

    PubMed Central

    Du, Yuhui; Song, Lifu; Feng, Wenjing; Pei, Guangsheng; Zheng, Ping; Yu, Zhichao; Sun, Jibin

    2013-01-01

    Lactococcus lactis subsp. lactis strain YF11 is a food preservative bacterium with a high capacity to produce nisin. Here, we announce the draft genome sequence of Lactococcus lactis subsp. lactis YF11 (2,527,433 bp with a G+C content of 34.81%). PMID:23929487

  12. Improved viability of bifidobacteria in fermented milk by cocultivation with Lactococcus lactis subspecies lactis.

    PubMed

    Odamaki, T; Xiao, J Z; Yonezawa, S; Yaeshima, T; Iwatsuki, K

    2011-03-01

    The poor survival of probiotic bacteria in commercial yogurts may limit their potential to exert health benefits in humans. The objective was to improve the survival of bifidobacteria in fermented milk. Cocultivation with some strains of Lactococcus lactis ssp. lactis improved the survival of bifidobacteria in fermented milk during refrigerated storage. Studies on one strain, Lc. lactis ssp. lactis MCC866, showed that the concentrations of dissolved oxygen were kept lower in the cocultivated fermented milk during storage compared with monocultured Bifidobacterium longum BB536 or samples cocultured with another noneffective Lc. lactis ssp. lactis strain. Degradation of genomic DNA was suppressed in the cocultivating system with Lc. lactis ssp. lactis MCC866. Several genes that participated in protection from active oxygen species (e.g., genes coding for alkyl hydroperoxide reductase and Fe(2+) transport system) were expressed at higher levels during refrigerated storage in Lc. lactis ssp. lactis MCC 866 compared with another noneffective Lc. lactis ssp. lactis strain. Concentration of free iron ion was also lower in supernatants of fermented milk cocultivated with B. longum BB536 and Lc. lactis ssp. lactis MCC866. These results suggest that Lc. lactis ssp. lactis MCC 866 is potentially superior in reducing oxygen damage and consequently improves the survival of bifidobacteria in the cocultivating system. This cocultivation system is of industrial interest for producing fermented milk containing viable bifidobacteria with long shelf life.

  13. Single-cell protein from methanol with Enterobacter aerogenes

    SciTech Connect

    Gnan, S.O.; Abodreheba, A.O.

    1987-02-20

    An identified Enterobacter aerogenes utilizing methanol as a sole carbon source was studied for the optimization of biomass production and the reduction of its nucleic acid content. Results indicated that the highest yield and conversion were obtained at 0.5% methanol. The addition of seawater as a source of trace elements has an adverse effect. However, the addition of urea as source of nitrogen enhanced the growth of E. aerogenes. Heat shock at 60 degrees C for one minute followed by incubation at 50 degrees C for 2 hours caused 72.6% reduction in the nucleic acid. 12 references.

  14. Enhancement of Nisin Production by Lactococcus lactis subsp. lactis.

    PubMed

    Dussault, Dominic; Vu, Khanh Dang; Lacroix, Monique

    2016-09-01

    Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.

  15. Single-electron aerogen bonds: Do they exist?

    NASA Astrophysics Data System (ADS)

    Esrafili, Mehdi D.; Mohammadian-Sabet, Fariba; Solimannejad, Mohammad

    2016-08-01

    A novel type of σ-hole interaction is characterized between some noble gas containing molecules (KrOF2, KrO3, XeOF2 and XeO3) and methyl (CH3) or ethyl (C2H5) radical by means of ab initio calculations. This interaction is named as single-electron aerogen bond (SEAB), in view of the concepts of aerogen bond and single-electron bond interactions. The properties of SEABs are studied by molecular electrostatic potential, quantum theory of atom in molecules, natural bonding orbital and noncovalent interaction index analyses. The formation of an O⋯H interaction tends to increase the strength of the SEAB, when they coexist in a ternary complex.

  16. Ribitol dehydrogenase from Klebsiella aerogenes. Purification and subunit structure

    PubMed Central

    Taylor, Susan S.; Rigby, Peter W. J.; Hartley, Brian S.

    1974-01-01

    Ribitol dehydrogenase has been purified to homogeneity from several strains of Klebsiella aerogenes. One strain yields 3–6g of pure enzyme from 1kg of cells. The enzyme is a tetramer of four subunits, mol.wt. 27000. Preliminary studies of the activity of the enzyme are reported. Peptide `maps' together with the amino acid composition indicate that the subunits are identical. ImagesPLATE 2PLATE 1 PMID:4618776

  17. Enterobacter aerogenes Needle Stick Leads to Improved Biological Management System

    SciTech Connect

    Johanson, Richard E.

    2004-08-01

    A laboratory worker who received a needle stick from a contaminated needle while working with a culture containing Enterobactor aerogenes developed a laboratory acquired infection. Although this organism has been shown to cause community and nosocomial infections, there have been no documented cases of a laboratory acquired infections. Lessons learned from the event led to corrective actions which included modification of lab procedures, development of a biological inventory tracking and risk identification system and the establishment of an effective biological safety program.

  18. BACT Simulation User Guide (Version 7.0)

    NASA Technical Reports Server (NTRS)

    Waszak, Martin R.

    1997-01-01

    This report documents the structure and operation of a simulation model of the Benchmark Active Control Technology (BACT) Wind-Tunnel Model. The BACT system was designed, built, and tested at NASA Langley Research Center as part of the Benchmark Models Program and was developed to perform wind-tunnel experiments to obtain benchmark quality data to validate computational fluid dynamics and computational aeroelasticity codes, to verify the accuracy of current aeroservoelasticity design and analysis tools, and to provide an active controls testbed for evaluating new and innovative control algorithms for flutter suppression and gust load alleviation. The BACT system has been especially valuable as a control system testbed.

  19. Molecular Analysis of Tetracycline Resistance in Pasteurella aerogenes

    PubMed Central

    Kehrenberg, Corinna; Schwarz, Stefan

    2001-01-01

    Tetracycline-resistant Pasteurella aerogenes isolates obtained from the intestinal tract of swine were investigated for their tet genes by PCR analysis and hybridization experiments. In contrast to Pasteurella isolates from the respiratory tract, tet(H) genes were detected in the chromosomal DNA of only 2 of the 24 isolates, one of which also carried two copies of a tet(B) gene. All other P. aerogenes isolates carried tet(B) genes, which are the predominant tet genes among Enterobacteriaceae. A single isolate harbored a tet(B) gene as part of a truncated Tn10 element on the 4.8-kb plasmid pPAT2. Comparative analysis of the pPAT2 sequence suggested that the Tn10 relic on plasmid pPAT2 is the result of several illegitimate recombination events. The remaining 21 P. aerogenes isolates carried one or two copies of the tet(B) gene in their chromosomal DNA. In the majority of the cases, these tet(B) genes were associated with copies of Tn10 as confirmed by their SfuI and BamHI hybridization patterns. No correlation between the number of tet gene copies and the MICs of tetracycline, doxycyline and minocycline was observed. PMID:11557485

  20. Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis LD61

    PubMed Central

    Falentin, Hélène; Naquin, Delphine; Loux, Valentin; Barloy-Hubler, Frédérique; Loubière, Pascal; Nouaille, Sébastien; Lavenier, Dominique; Le Bourgeois, Pascal; François, Patrice; Schrenzel, Jacques; Hernandez, David; Even, Sergine

    2014-01-01

    Lactococcus lactis is widely used in the dairy industry. We report the draft genome sequence of L. lactis subsp. lactis bv. diacetylactis LD61, an industrial and extensively studied strain. In contrast to the closely related and plasmidless strain IL1403, LD61 contains 6 plasmids, and the genome sequence provides additional information related to adaptation to the dairy environment. PMID:24435871

  1. Biological Conversion of Glycerol to Ethanol by Enterobacter aerogenes

    NASA Astrophysics Data System (ADS)

    Nwachukwu, Raymond E. S.

    In a search to turn the economically and environmentally non-valuable "waste" streams of biodiesel production into a profitable byproduct, a mutant strain of Enterobacter aerogenes ATCC 13048 was developed by six-tube subculturing technique. This technique is based on the principle of adaptive evolution, and involved subculturing the bacterium in a tryptic soy broth without dextrose (TSB) containing specific glycerol and ethanol concentration for six consecutive times. Then, the six consecutive subculturing was repeated in a fresh TSB of higher glycerol and ethanol concentrations. A new mutant strain, E. aerogenes S012, which could withstand a combination of 200 g/l glycerol and 30 g/l ethanol concentrations, was developed. The wild and mutant strains were used for the fermentation of pure (P-) and recovered (R-) glycerol. Taguchi and full factorial methods of design of experiments were used to screen and optimize the important process factors that influence the microbial production of ethanol. A statistically sound regression model was used to establish the mathematical relationship between the process variables and ethanol production. Temperature of 38°C, agitation speed of 200 rpm, pH of 6.3-6.6, and microaerobic condition were the optimum process conditions. Different pretreatment methods to recover glycerol from the crude glycerol and the subsequent fermentation method showed that direct acidification using 85% H3PO4 was the best. The R-glycerol contained 51% pure glycerol and 21% methanol. The wild strain, E. aerogenes ATCC 13048, produced only 12 g/l and 12.8 g/l ethanol from 20 g/l P- and R-glycerol respectively, and could not utilize higher glycerol concentrations. The mutant, E. aerogenes S012, produced ethanol amount and yield of 43 g/l and 1.12 mol/mol-glycerol from P-glycerol, respectively within 96 h. It also produced ethanol amount and yield of 26.8 g/l and 1.07 mol/mol-glycerol, respectively, from R-glycerol within the same duration. In a

  2. Microbiota of Minas cheese as influenced by the nisin producer Lactococcus lactis subsp. lactis GLc05.

    PubMed

    Perin, Luana Martins; Dal Bello, Barbara; Belviso, Simona; Zeppa, Giuseppe; de Carvalho, Antônio Fernandes; Cocolin, Luca; Nero, Luís Augusto

    2015-12-01

    Minas cheese is a popular dairy product in Brazil that is traditionally produced using raw or pasteurized cow milk. This study proposed an alternative production of Minas cheese using raw goat milk added of a nisin producer Lactococcus lactis subsp. lactis GLc05. An in situ investigation was carried on to evaluate the interactions between the L. lactis subsp. lactis GLc05 and the autochthonous microbiota of a Minas cheese during the ripening; production of biogenic amines (BAs) was assessed as a safety aspect. Minas cheese was produced in two treatments (A, by adding L. lactis subsp. lactis GLc05, and B, without adding this strain), in three independent repetitions (R1, R2, and R3). Culture dependent (direct plating) and independent (rep-PCR and PCR-DGGE) methods were employed to characterize the microbiota and to assess the possible interferences caused by L. lactis subsp. lactis GLc05. BA amounts were measured using HPLC. A significant decrease in coagulase-positive cocci was observed in the cheeses produced by adding L. lactis subsp. lactis GLc05 (cheese A). The rep-PCR and PCR-DGGE highlighted the differences in the microbiota of both cheeses, separating them into two different clusters. Lactococcus sp. was found as the main microorganism in both cheeses, and the microbiota of cheese A presented a higher number of species. High concentrations of tyramine were found in both cheeses and, at specific ripening times, the BA amounts in cheese B were significantly higher than in cheese A (p<0.05). The interaction of nisin producer L. lactis subsp. lactis GLc05 was demonstrated in situ, by demonstration of its influence in the complex microbiota naturally present in a raw goat milk cheese and by controlling the growth of coagulase-positive cocci. L. lactis subsp. lactis GLc05 influenced also the production of BA determining that their amounts in the cheeses were maintained at acceptable levels for human consumption.

  3. Microbiota of Minas cheese as influenced by the nisin producer Lactococcus lactis subsp. lactis GLc05.

    PubMed

    Perin, Luana Martins; Dal Bello, Barbara; Belviso, Simona; Zeppa, Giuseppe; de Carvalho, Antônio Fernandes; Cocolin, Luca; Nero, Luís Augusto

    2015-12-01

    Minas cheese is a popular dairy product in Brazil that is traditionally produced using raw or pasteurized cow milk. This study proposed an alternative production of Minas cheese using raw goat milk added of a nisin producer Lactococcus lactis subsp. lactis GLc05. An in situ investigation was carried on to evaluate the interactions between the L. lactis subsp. lactis GLc05 and the autochthonous microbiota of a Minas cheese during the ripening; production of biogenic amines (BAs) was assessed as a safety aspect. Minas cheese was produced in two treatments (A, by adding L. lactis subsp. lactis GLc05, and B, without adding this strain), in three independent repetitions (R1, R2, and R3). Culture dependent (direct plating) and independent (rep-PCR and PCR-DGGE) methods were employed to characterize the microbiota and to assess the possible interferences caused by L. lactis subsp. lactis GLc05. BA amounts were measured using HPLC. A significant decrease in coagulase-positive cocci was observed in the cheeses produced by adding L. lactis subsp. lactis GLc05 (cheese A). The rep-PCR and PCR-DGGE highlighted the differences in the microbiota of both cheeses, separating them into two different clusters. Lactococcus sp. was found as the main microorganism in both cheeses, and the microbiota of cheese A presented a higher number of species. High concentrations of tyramine were found in both cheeses and, at specific ripening times, the BA amounts in cheese B were significantly higher than in cheese A (p<0.05). The interaction of nisin producer L. lactis subsp. lactis GLc05 was demonstrated in situ, by demonstration of its influence in the complex microbiota naturally present in a raw goat milk cheese and by controlling the growth of coagulase-positive cocci. L. lactis subsp. lactis GLc05 influenced also the production of BA determining that their amounts in the cheeses were maintained at acceptable levels for human consumption. PMID:26310130

  4. Regulation of Nitrate Assimilation and Nitrate Respiration in Aerobacter aerogenes

    PubMed Central

    Van 'T Riet, J.; Stouthamer, A. H.; Planta, R. J.

    1968-01-01

    The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH4+ to aerobiosis in the same medium, but without NH4+, the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH4+, under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein. PMID:5726295

  5. Hydrogen production from biodiesel byproduct by immobilized Enterobacter aerogenes.

    PubMed

    Han, Jinmi; Lee, Dohoon; Cho, Jinku; Lee, Jeewon; Kim, Sangyong

    2012-01-01

    The recent rapid growth of the biodiesel industry has generated a significant amount of glycerol as a byproduct. As a result, the price of glycerol is currently relatively low, making it an attractive starting material for the production of chemicals with higher values. Crude glycerol can be directly converted through microbial fermentation into various chemicals such as hydrogen. In this study, we optimized immobilization of a facultative hydrogen producing microorganism, Enterobacter aerogenes, with the goal of developing biocatalysts that was appropriate for the continuous hydrogen production from glycerol. Several carriers were tested and agar was found to be the most effective. In addition, it was clearly shown that variables such as the carrier content and cell loading should be controlled for the immobilization of biocatalysts with high hydrogen productivity, stability, and reusability. After optimization of these variables, we were able to obtain reusable biocatalysts that could directly convert the byproduct stream from biodiesel processes into hydrogen in continuous processes.

  6. Isolation and characterization of a bacteriophage phiEap-2 infecting multidrug resistant Enterobacter aerogenes.

    PubMed

    Li, Erna; Wei, Xiao; Ma, Yanyan; Yin, Zhe; Li, Huan; Lin, Weishi; Wang, Xuesong; Li, Chao; Shen, Zhiqiang; Zhao, Ruixiang; Yang, Huiying; Jiang, Aimin; Yang, Wenhui; Yuan, Jing; Zhao, Xiangna

    2016-01-01

    Enterobacter aerogenes (Enterobacteriaceae) is an important opportunistic pathogen that causes hospital-acquired pneumonia, bacteremia, and urinary tract infections. Recently, multidrug-resistant E. aerogenes have been a public health problem. To develop an effective antimicrobial agent, bacteriophage phiEap-2 was isolated from sewage and its genome was sequenced because of its ability to lyse the multidrug-resistant clinical E. aerogenes strain 3-SP. Morphological observations suggested that the phage belongs to the Siphoviridae family. Comparative genome analysis revealed that phage phiEap-2 is related to the Salmonella phage FSL SP-031 (KC139518). All of the structural gene products (except capsid protein) encoded by phiEap-2 had orthologous gene products in FSL SP-031 and Serratia phage Eta (KC460990). Here, we report the complete genome sequence of phiEap-2 and major findings from the genomic analysis. Knowledge of this phage might be helpful for developing therapeutic strategies against E. aerogenes. PMID:27320081

  7. Isolation and characterization of a bacteriophage phiEap-2 infecting multidrug resistant Enterobacter aerogenes.

    PubMed

    Li, Erna; Wei, Xiao; Ma, Yanyan; Yin, Zhe; Li, Huan; Lin, Weishi; Wang, Xuesong; Li, Chao; Shen, Zhiqiang; Zhao, Ruixiang; Yang, Huiying; Jiang, Aimin; Yang, Wenhui; Yuan, Jing; Zhao, Xiangna

    2016-06-20

    Enterobacter aerogenes (Enterobacteriaceae) is an important opportunistic pathogen that causes hospital-acquired pneumonia, bacteremia, and urinary tract infections. Recently, multidrug-resistant E. aerogenes have been a public health problem. To develop an effective antimicrobial agent, bacteriophage phiEap-2 was isolated from sewage and its genome was sequenced because of its ability to lyse the multidrug-resistant clinical E. aerogenes strain 3-SP. Morphological observations suggested that the phage belongs to the Siphoviridae family. Comparative genome analysis revealed that phage phiEap-2 is related to the Salmonella phage FSL SP-031 (KC139518). All of the structural gene products (except capsid protein) encoded by phiEap-2 had orthologous gene products in FSL SP-031 and Serratia phage Eta (KC460990). Here, we report the complete genome sequence of phiEap-2 and major findings from the genomic analysis. Knowledge of this phage might be helpful for developing therapeutic strategies against E. aerogenes.

  8. TEM Derivative-Producing Enterobacter aerogenes Strains: Dissemination of a Prevalent Clone

    PubMed Central

    Dumarche, P.; De Champs, C.; Sirot, D.; Chanal, C.; Bonnet, R.; Sirot, J.

    2002-01-01

    TEM-24 (CAZ-6) extended-spectrum β-lactamase (ESBL) was detected in 1988 in Clermont-Ferrand, France, in Klebsiella pneumoniae (blaTEM-24) and Enterobacter aerogenes (blaTEM-24b), and since 1994, a TEM-24-producing E. aerogenes clonal strain has been observed elsewhere in the country. To determine if the spread of this clonal strain was restricted to TEM-24-producing E. aerogenes strains, 84 E. aerogenes strains (non-TEM/SHV-producing strains, TEM-1- or -2-producing strains, and different ESBL-producing strains), isolated from 1988 to 1999 in Clermont-Ferrand (n = 59) and in 11 other French hospitals in 1998 (n = 25), were studied. A clonal strain was found for TEM-24- but also for TEM-3- and TEM-1- or 2-producing isolates. This study shows that there is a clonal strain dependent on acquisition of the TEM-type enzyme (TEM-24 and other TEM types). PMID:11897606

  9. Isolation and characterization of a bacteriophage phiEap-2 infecting multidrug resistant Enterobacter aerogenes

    PubMed Central

    Li, Erna; Wei, Xiao; Ma, Yanyan; Yin, Zhe; Li, Huan; Lin, Weishi; Wang, Xuesong; Li, Chao; Shen, Zhiqiang; Zhao, Ruixiang; Yang, Huiying; Jiang, Aimin; Yang, Wenhui; Yuan, Jing; Zhao, Xiangna

    2016-01-01

    Enterobacter aerogenes (Enterobacteriaceae) is an important opportunistic pathogen that causes hospital-acquired pneumonia, bacteremia, and urinary tract infections. Recently, multidrug-resistant E. aerogenes have been a public health problem. To develop an effective antimicrobial agent, bacteriophage phiEap-2 was isolated from sewage and its genome was sequenced because of its ability to lyse the multidrug-resistant clinical E. aerogenes strain 3-SP. Morphological observations suggested that the phage belongs to the Siphoviridae family. Comparative genome analysis revealed that phage phiEap-2 is related to the Salmonella phage FSL SP-031 (KC139518). All of the structural gene products (except capsid protein) encoded by phiEap-2 had orthologous gene products in FSL SP-031 and Serratia phage Eta (KC460990). Here, we report the complete genome sequence of phiEap-2 and major findings from the genomic analysis. Knowledge of this phage might be helpful for developing therapeutic strategies against E. aerogenes. PMID:27320081

  10. Complete Genome Sequence of Lactococcus lactis subsp. lactis A12, a Strain Isolated from Wheat Sourdough.

    PubMed

    Guellerin, Maéva; Passerini, Delphine; Fontagné-Faucher, Catherine; Robert, Hervé; Gabriel, Valérie; Loux, Valentin; Klopp, Christophe; Le Loir, Yves; Coddeville, Michèle; Daveran-Mingot, Marie-Line; Ritzenthaler, Paul; Le Bourgeois, Pascal

    2016-01-01

    We report here the complete genome sequence of Lactococcus lactis subsp. lactis strain A12, a strain isolated from sourdough. The circular chromosome and the four plasmids reveal genes involved in carbohydrate metabolism that are potentially required for the persistence of this strain in such a complex ecosystem. PMID:27634985

  11. Complete Genome Sequence of Lactococcus lactis subsp. lactis A12, a Strain Isolated from Wheat Sourdough

    PubMed Central

    Guellerin, Maéva; Passerini, Delphine; Fontagné-Faucher, Catherine; Robert, Hervé; Gabriel, Valérie; Loux, Valentin; Klopp, Christophe; Le Loir, Yves; Coddeville, Michèle; Daveran-Mingot, Marie-Line; Ritzenthaler, Paul

    2016-01-01

    We report here the complete genome sequence of Lactococcus lactis subsp. lactis strain A12, a strain isolated from sourdough. The circular chromosome and the four plasmids reveal genes involved in carbohydrate metabolism that are potentially required for the persistence of this strain in such a complex ecosystem. PMID:27634985

  12. Draft Genome Sequence of the Putrescine-Producing Strain Lactococcus lactis subsp. lactis 1AA59

    PubMed Central

    del Rio, Beatriz; Linares, Daniel M.; Fernandez, María; Mayo, Baltasar; Martín, M. Cruz

    2015-01-01

    We report here the 2,576,542-bp genome annotated draft assembly sequence of Lactococcus lactis subsp. lactis 1AA59. This strain—isolated from a traditional cheese—produces putrescine, one of the most frequently biogenic amines found in dairy products. PMID:26089428

  13. Complete Genome Sequence of Lactococcus lactis subsp. lactis A12, a Strain Isolated from Wheat Sourdough.

    PubMed

    Guellerin, Maéva; Passerini, Delphine; Fontagné-Faucher, Catherine; Robert, Hervé; Gabriel, Valérie; Loux, Valentin; Klopp, Christophe; Le Loir, Yves; Coddeville, Michèle; Daveran-Mingot, Marie-Line; Ritzenthaler, Paul; Le Bourgeois, Pascal

    2016-09-15

    We report here the complete genome sequence of Lactococcus lactis subsp. lactis strain A12, a strain isolated from sourdough. The circular chromosome and the four plasmids reveal genes involved in carbohydrate metabolism that are potentially required for the persistence of this strain in such a complex ecosystem.

  14. Comparative Phenotypic and Molecular Genetic Profiling of Wild Lactococcus lactis subsp. lactis Strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris Genotypes, Isolated from Starter-Free Cheeses Made of Raw Milk▿

    PubMed Central

    Fernández, Elena; Alegría, Ángel; Delgado, Susana; Martín, M. Cruz; Mayo, Baltasar

    2011-01-01

    Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies

  15. BACT analysis under the Clean Air Act's PCD program

    SciTech Connect

    Simms, P.; Walke, J.

    2006-11-15

    Before a company may build a new major industrial source of air pollution, or make modifications to an existing major source in the USA it must apply for and receive a Clean Air Act (CAA) Prevention of Significant Deterioration (PSD) permit. State environmental agencies typically issue such permits, either under state law or by exercising delegated authority to implement the federal PSD program. To fully comply with the CAA, the emissions limits identified as BACT must incorporate consideration of more than just add-on emissions control technology, they must also reflect appropriate considerations of fuel quality (e.g. low-sulfur coal) and process changes (e.g. advanced combustion techniques) as a means of controlling emissions, and must consider the other environmental and public welfare benefits of the identified emissions control options. Several states including New Mexico and Illinois have already determined that innovated technologies, such as Integrated Gasification Combined Cycle (IGCC), must be considered in connection with the BACT analysis for new coal-fired power plants. Even the notion that BACT is categorically limited in scope to the general type of facility proposed is contrary to EPA precedent. For example, the Environmental Appeals Board (EAB) has explained that permitting authorities retain the discretion under the definition of BACT to require dramatically different facility designs (e.g. a natural gas plant instead of a coal-fired power plant). The best advice for any permit applicant is to include in the BACT analysis a careful and honest examination of better performing alternative processes and/or innovative combustion techniques and to aggressively pursue such options wherever feasible. 17 refs.

  16. Oral delivery of beta-lactamase by Lactococcus lactis subsp. lactis transformed with Plasmid ss80.

    PubMed

    Kaushal, Gagan; Shao, Jun

    2006-04-01

    The objective was to use normal flora to deliver protein/peptide drugs orally. A probiotic bacterium, Lactococcus lactis subsp. lactis (L. lactis) transformed with Plasmid ss80, which made it able to synthesize and secrete beta-lactamase, a 29 kDa protein, was used as the delivery system for beta-lactamase. Oral absorption of beta-lactamase in rats when delivered by this L. lactis system was investigated. The oral bioavailability of beta-lactamase delivered by 3x10(7) of the L. lactis was equivalent to 209 mU of i.v. dose, and the estimated relative bioavailability was 16.7%. When delivered by beta-lactamase free solution form, the relative oral bioavailability was 4.7%, which increased to 6.0% when co-administered with 3x10(7) of the untransformed L. lactis. The results demonstrated that the L. lactis significantly increased the beta-lactamase oral bioavailability by 2-3-folds (p<0.01), the mean residence time (MRT) by 3-4 times (p<0.01), and the mean absorption time (MAT) by 6-14 times (p<0.01), as compared to the free solution form with/without the untransformed L. lactis. In conclusion, the L. lactis is more efficient in delivering beta-lactamase orally compared with the free solution form. It also provides a sustained delivery mechanism for beta-lactamase. Gene-transformed normal flora may be used as an efficient and sustained delivery system for protein drugs through oral route.

  17. VetBact - culturing bacteriological knowledge for veterinarians.

    PubMed

    Johansson, Karl-Erik

    2014-02-15

    In 2004, Karl-Erik Johansson, then professor of veterinary bacteriology at the veterinary school at Uppsala in Sweden, was asked by his students for a list of the most important bacteria and the diseases that they cause. So began the development of VetBact, an online database giving details of the bacterial species with most relevance to veterinary medicine. The non-commercial database, www.vetbact.org, has since grown and can now be accessed by veterinarians and others worldwide.

  18. An efficient ribitol-specific dehydrogenase from Enterobacter aerogenes.

    PubMed

    Singh, Ranjitha; Singh, Raushan; Kim, In-Won; Sigdel, Sujan; Kalia, Vipin C; Kang, Yun Chan; Lee, Jung-Kul

    2015-05-01

    An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity.

  19. Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain

    PubMed Central

    Zuljan, Federico; Espariz, Martín; Blancato, Victor S.; Esteban, Luis; Alarcón, Sergio

    2016-01-01

    We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains. PMID:26847906

  20. Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain.

    PubMed

    Zuljan, Federico; Espariz, Martín; Blancato, Victor S; Esteban, Luis; Alarcón, Sergio; Magni, Christian

    2016-02-04

    We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains.

  1. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality. PMID:25763065

  2. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Characterization of the bacteriocin

    PubMed Central

    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality. PMID:25763065

  3. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.

  4. Comparison of the clinical and microbiologic characteristics of patients with Enterobacter cloacae and Enterobacter aerogenes bacteremia: a prospective observation study.

    PubMed

    Song, Eun Hee; Park, Ki-Ho; Jang, Eun-Young; Lee, Eun Jung; Chong, Yong Pil; Cho, Oh-Hyun; Kim, Sung-Han; Lee, Sang-Oh; Sung, Heungsup; Kim, Mi-Na; Jeong, Jin-Yong; Kim, Yang Soo; Woo, Jun Hee; Choi, Sang-Ho

    2010-04-01

    We compared the characteristics and outcomes of 172 Enterobacter cloacae bacteremia and 67 Enterobacter aerogenes bacteremia (EAB) cases. Antimicrobial resistance rates to E. cloacae were higher than those to E. aerogenes. However, EAB more frequently presented as septic shock and was associated with poorer outcomes.

  5. The melatonin-sensitive circadian clock of the enteric bacterium Enterobacter aerogenes.

    PubMed

    Paulose, Jiffin K; Cassone, Vincent M

    2016-09-01

    Circadian clocks are fundamental properties of all eukaryotic organisms and at least some prokaryotic organisms. Recent studies in our laboratory have shown that the gastrointestinal system contains a circadian clock that controls many, if not all, aspects of gastrointestinal function. We now report that at least one species of intestinal bacteria, Enterobacter aerogenes, responds to the pineal and gastrointestinal hormone melatonin by an increase in swarming activity. This swarming behavior is expressed rhythmically, with a period of approximately 24 hrs. Transformation of E. aerogenes to express luciferase with a MotA promoter reveals circadian patterns of bioluminescence that are synchronized by melatonin and whose periods are temperature compensated from 26°C to 40°C. Bioinformatics suggest similarities between the E. aerogenes and cyanobacterial clocks, suggesting the circadian clock may have evolved very early in the evolution of life. They also point to a coordination of host circadian clocks with those residing in the microbiota themselves. PMID:27387841

  6. The melatonin-sensitive circadian clock of the enteric bacterium Enterobacter aerogenes.

    PubMed

    Paulose, Jiffin K; Cassone, Vincent M

    2016-09-01

    Circadian clocks are fundamental properties of all eukaryotic organisms and at least some prokaryotic organisms. Recent studies in our laboratory have shown that the gastrointestinal system contains a circadian clock that controls many, if not all, aspects of gastrointestinal function. We now report that at least one species of intestinal bacteria, Enterobacter aerogenes, responds to the pineal and gastrointestinal hormone melatonin by an increase in swarming activity. This swarming behavior is expressed rhythmically, with a period of approximately 24 hrs. Transformation of E. aerogenes to express luciferase with a MotA promoter reveals circadian patterns of bioluminescence that are synchronized by melatonin and whose periods are temperature compensated from 26°C to 40°C. Bioinformatics suggest similarities between the E. aerogenes and cyanobacterial clocks, suggesting the circadian clock may have evolved very early in the evolution of life. They also point to a coordination of host circadian clocks with those residing in the microbiota themselves.

  7. Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes.

    PubMed

    Verthé, Kristof; Verstraete, Willy

    2006-09-01

    In this study, the use of flow cytometry to analyze phage-mediated killing of Enterobacter aerogenes under varying conditions of temperature and nutrient availability was assessed. Bacteriophage UZ1, specific for an E. aerogenes strain, was applied at a multiplicity of infection (MOI) of 1 and 1000 to a Teflon surface, artificially infected with its host at a level of 4.5 log cells. After incubation for 20 h, bacteriophages were quantified using the soft agar layer method. For the quantification of bacterial cells, plate counting and flow cytometric analysis of live/dead stained cells were performed in parallel. At an MOI of 1, phage treatment was successful only after incubation under nutrient-rich conditions at 37 degrees C: E. aerogenes cells were not detected and a tenfold increase in phage UZ1 was observed. At a MOI of 1000, no E. aerogenes cells could be cultured after incubation at 37 and 4 degrees C. However, flow cytometric analysis revealed that lysis did not occur at 4 degrees C but was achieved during subsequent plate culture. In conclusion, the use of flow cytometry enabled identification of culture-based bias during plate culture. The flow cytometric assay used in this study proved to be rapid, as this culture-independent method does not require lengthy incubation periods post-sampling. The bacteriophage-mediated killing of E. aerogenes cells on Teflon surfaces indicated that disinfection of E. aerogenes with bacteriophage UZ1 can be successful when high MOIs are achieved, while at low multiplicities of infection conditions favorable for phage replication are required.

  8. Inorganic phosphate accumulation and cadmium detoxification in Klebsiella aerogenes NCTC 418 growing in continuous culture

    SciTech Connect

    Aiking, H.; Stijnman, A.; van Garderen, C.; van Heerikhuizen, H.; van Riet, J.

    1984-02-01

    Klebsiella aerogenes NCTC-418, growing in the presence of cadmium under glucose-, sulfate-, or phosphate-limited conditions in continuous culture, exhibits two different cadmium detoxifying mechanisms. In addition to sulfide formation, increased accumulation of P/sub i/ is demonstrated as a novel mechanism. Intracellular cadmium is always quantitatively counterbalanced by a concerted increase in both inorganic sulfide and P/sub i/ contents of the cells. This led to the conclusion that production of sulfide and accumulation of P/sub i/ are detoxification mechanisms present in K. aerogenes but that their relative importance is crucially dependent on the strain and the growth conditions employed.

  9. Complete genome sequence of Bifidobacterium animalis subsp. lactis BLC1.

    PubMed

    Bottacini, Francesca; Dal Bello, Fabio; Turroni, Francesca; Milani, Christian; Duranti, Sabrina; Foroni, Elena; Viappiani, Alice; Strati, Francesco; Mora, Diego; van Sinderen, Douwe; Ventura, Marco

    2011-11-01

    Bifidobacterium animalis subsp. lactis BLC1 is a probiotic bacterium that is widely exploited by food industries as the active ingredient of various functional foods. Here we report the complete genome sequence of B. animalis subsp. lactis BLC1, which is expected to provide insights into the biology of this health-promoting microorganism and improve our understanding of its phylogenetic relatedness with other members of the B. animalis subsp. lactis taxon. PMID:22038957

  10. Novel Antibacterial Activity of Lactococcus Lactis Subspecies Lactis Z11 Isolated from Zabady

    PubMed Central

    Enan, Gamal; Abdel-Shafi, Seham; Ouda, Sahar; Negm, Sally

    2013-01-01

    The purpose of this study was to select and characterize a probiotic bacterium with distinctive antimicrobial activities. In this respect, Lactococcus lactis subspecies lactis Z11 (L. lactis Z11) isolated from Zabady (Arabian yoghurt) inhibited other strains of lactic acid bacteria and some food-born pathogens including Listeria monocytogenes, Bacillus cereus and staphylococcus aureus. The inhibitory activity of cell free supernatant (CFS) of L. lactis Z11 isolated from zabady was lost by proteolytic enzymes, heat resistant. Consequently, the active substance(s) of CFS was characterized as a bacteriocin. This bacteriocin has been shown to consist of protein but has no lipidic or glucidic moieties in its active molecule. Its activity was stable in the pH range 2.0 to 7.0 and was not affected by organic solvents. The L. lactis Z11 bacteriocin was produced in CFS throughout the mide to the late exponential phase of growth of the producer organism and maximum bacteriocin production was obtained at initial pH 6.5 at incubation temperature of about 30°C. PMID:24151453

  11. Benchmark Active Controls Technology (BACT) Wing CFD Results

    NASA Technical Reports Server (NTRS)

    Schuster, David M.; Bartels, Robert E.

    2000-01-01

    The Benchmark Active Controls Technology (BACT) wing test (see chapter 8E) provides data for the validation of aerodynamic, aeroelastic, and active aeroelastic control simulation codes. These data provide a rich database for development and validation of computational aeroelastic and aeroservoelastic methods. In this vein, high-level viscous CFD analyses of the BACT wing have been performed for a subset of the test conditions available in the dataset. The computations presented in this section investigate the aerodynamic characteristics of the rigid clean wing configuration as well as simulations of the wing with a static and oscillating aileron and spoiler deflection. Two computational aeroelasticity codes extensively used at NASA Langley Research Center are implemented in this simulation. They are the ENS3DAE and CFL3DAE computational aeroelasticity programs. Both of these methods solve the three-dimensional compressible Navier-Stokes equations for both rigid and flexible vehicles, but they use significantly different approaches to the solution 6f the aerodynamic equations of motion. Detailed descriptions of both methods are presented in the following section.

  12. Induction by Klebsiella aerogenes of a Melanin-Like Pigment in Cryptococcus neoformans

    PubMed Central

    Frases, Susana; Chaskes, Stuart; Dadachova, Ekaterina; Casadevall, Arturo

    2006-01-01

    While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37°C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment. PMID:16461709

  13. Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans.

    PubMed

    Frases, Susana; Chaskes, Stuart; Dadachova, Ekaterina; Casadevall, Arturo

    2006-02-01

    While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.

  14. Development of Enterobacter aerogenes fuel cells: from in situ biohydrogen oxidization to direct electroactive biofilm.

    PubMed

    Zhuang, Li; Zhou, Shungui; Yuan, Yong; Liu, Tinglin; Wu, Zhifeng; Cheng, Jiong

    2011-01-01

    This study described an Enterobacter aerogenes-catalyzed microbial fuel cell (MFC) with a carbon-based anode that exhibited a maximum power density of 2.51 W/m(3) in the absence of artificial electron mediators. The MFC was started up rapidly, within hours, and the current generation in the early stage was demonstrated to result from in situ oxidation of biohydrogen produced by E. aerogenes during glucose fermentation. Over periodic replacement of substrate, both planktonic biomass in the culture liquid and hydrogen productivity decreased, while increased power density and coulombic efficiency and decreased internal resistance were unexpectedly observed. Using scanning electron microscopy and cyclic voltammetry, it was found that the enhanced MFC performance was associated with the development of electroactive biofilm on the anodic surface, proposed to involve an acclimation and selection process of E. aerogenes cells under electrochemical tension. The significant advantage of rapid start-up and the ability to develop an electroactive biofilm identifies E. aerogenes as a suitable biocatalyst for MFC applications.

  15. An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates.

    PubMed

    Lavigne, Jean-Philippe; Sotto, Albert; Nicolas-Chanoine, Marie-Hélène; Bouziges, Nicole; Pagès, Jean-Marie; Davin-Regli, Anne

    2013-02-01

    Imipenem (IPM) is a carbapenem antibiotic frequently used in severe hospital infections. Several reports have mentioned the emergence of resistant isolates exhibiting membrane modifications. A study was conducted between September 2005 and August 2007 to survey infections due to Enterobacter aerogenes in patients hospitalised in a French university hospital. Resistant E. aerogenes clinical isolates obtained from patients treated with IPM and collected during the 3 months following initiation of treatment were phenotypically and molecularly characterised for β-lactamases, efflux pumps activity and outer membrane proteins. Among the 339 patients infected with E. aerogenes during the study period, 41 isolates (12.1%) were resistant to extended-spectrum cephalosporins and 17 patients (5.0%) were treated with IPM. The isolates from these 17 patients presented TEM-24 and basal efflux expression. Following IPM treatment, an IPM-intermediate-susceptible (IPM-I) isolate emerged in 11 patients and an IPM-resistant (IPM-R) isolate in 6 patients. A change in the porin balance (Omp35/Omp36) was observed in IPM-I isolates exhibiting ertapenem resistance. Finally, a porin deficiency (Omp35 and Omp36 absence) was detected in IPM-R isolates associated with efflux pump expression. This study indicates that the alteration in porin expression, including the shift of porin expression and lack of porins, contribute to the E. aerogenes adaptive response to IPM treatment.

  16. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: evaluation of the probiotic potential.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties.

  17. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: evaluation of the probiotic potential.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties. PMID:25477942

  18. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Evaluation of the probiotic potential

    PubMed Central

    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2014-01-01

    Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties. PMID:25477942

  19. Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.

    PubMed

    Zhu, Duolong; Liu, Fulu; Xu, Haijin; Bai, Yanling; Zhang, Xiuming; Saris, Per Erik Joakim; Qiao, Mingqiang

    2015-08-01

    The synthesis of heterologous proteins in Lactococcus lactis is strongly influenced by the promoter selected for the expression. The nisin A promoter is commonly used for induced expression of proteins in L. lactis, whereas few constitutive promoters (P45 and the weaker P32) have been used for protein expression studies. In this study, eight different putative strong constitutive promoters were identified through transcriptional analysis of L. lactis N8 and were investigated for their capability to drive nisZ gene expression with promoters P45 and P32 as control. Four strong promoters (P8, P5, P3 and P2) were identified as having a transcriptional activity that was higher than that of P45 through RT-qPCR and agar-diffusion experiments. In addition, these four promoters were fused to the erythromycin resistant gene (ermC) with promoter P45 as control and inserted into the backbone of the pNZ8048 vector. The transcriptional efficiencies of promoters P8, P5, P2 and P3 were all higher than promoter P45 based on the obtained MIC50 values and they all showed different activity levels. In conclusion, four strong constitutive promoters with a wide range of promoter activities were identified and are suitable for protein production in L. lactis. PMID:26156144

  20. A Deficiency in Aspartate Biosynthesis in Lactococcus lactis subsp. lactis C2 Causes Slow Milk Coagulation†

    PubMed Central

    Wang, Hua; Yu, Weizhu; Coolbear, Tim; O’Sullivan, Dan; McKay, Larry L.

    1998-01-01

    A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc−) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis. PMID:9572935

  1. Bactériophages et phagothérapie: utilisation de virus naturels pour traiter les infections bactériennes

    PubMed Central

    Ravat, F.; Jault, P.; Gabard, J.

    2015-01-01

    Summary L’utilisation des bactériophages, prédateurs naturels des bactéries, est une technique pionnière efficace de lutte contre les infections bactériennes. Tombée dans l’oubli depuis un demi-siècle du coté occidental de l’ex-rideau de fer, elle fait toujours partie de l’arsenal thérapeutique des pays de l’ex-Europe de l’Est, au point de constituer une arme de choix dans la politique de santé publique de ces pays. l’émergence de bactéries multirésistantes et le risque de revenir à l’ère pré-antibiotique ont fait ressortir la phagothérapie de l’oubli injuste auquel elle avait été confinée. la biologie et la place du bactériophage dans la nature sont décrites ici. les tenants et les aboutissants de la phagothérapie et les conditions de son retour sur le devant de la scène sont explicitées. PMID:26668557

  2. Detection and viability of Lactococcus lactis throughout cheese ripening.

    PubMed

    Ruggirello, Marianna; Dolci, Paola; Cocolin, Luca

    2014-01-01

    Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.

  3. Detection and Viability of Lactococcus lactis throughout Cheese Ripening

    PubMed Central

    Cocolin, Luca

    2014-01-01

    Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese. PMID:25503474

  4. Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes.

    PubMed

    Mukherjee, J; Majumdar, S; Scheper, T

    2000-02-01

    The carbon and nitrogen sources most suitable for L-asparaginase production by Enterobacter aerogenes were selected and their concentrations optimized in shake-flask cultures. Sodium citrate (1.0%) and diammonium hydrogen phosphate (0.16%) proved to be the best sources of carbon and nitrogen, respectively. Nitrogen catabolite repression of enzyme formation was absent in this bacterium. Cultivation in a reactor showed that the dissolved oxygen level is the limiting factor for L-asparaginase production by E. aerogenes. Glucose was found to be a repressor of enzyme synthesis. Asparagine was absent intracellularly when the L-asparaginase level was high. An increase in the extracellular alanine level when the dissolved oxygen remained low indicated a shift from aerobic to fermentative metabolism. PMID:10709980

  5. Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes.

    PubMed

    Lin, Richen; Cheng, Jun; Ding, Lingkan; Song, Wenlu; Liu, Min; Zhou, Junhu; Cen, Kefa

    2016-05-01

    Ferric oxide nanoparticles (FONPs) were used to facilitate dark hydrogen fermentation using Enterobacter aerogenes. The hydrogen yield of glucose increased from 164.5±2.29 to 192.4±1.14mL/g when FONPs concentration increased from 0 to 200mg/L. SEM images of E. aerogenes demonstrated the existence of bacterial nanowire among cells, suggesting FONPs served as electron conduits to enhance electron transfer. TEM showed cellular internalization of FONPs, indicating hydrogenase synthesis and activity was potentially promoted due to the released iron element. When further increasing FONPs concentration to 400mg/L, the hydrogen yield of glucose decreased to 147.2±2.54mL/g. Soluble metabolic products revealed FONPs enhanced acetate pathway of hydrogen production, but weakened ethanol pathway. This shift of metabolic pathways allowed more nicotinamide adenine dinucleotide for reducing proton to hydrogen.

  6. Is Aerogen-π Interaction Capable of Initiating the Noncovalent Chemistry of Group 18?

    PubMed

    Miao, Junjian; Song, Bo; Gao, Yi

    2015-12-01

    The interactions between atoms of noble gases and π systems are generally considered as van der Waals interaction, which have not attracted attention yet. Herein, we present high-level ab initio calculations to show the unexpected noncovalent interaction between a covalently bonded noble gas atom and a delocalized aromatic π electron using XeO3⋅benzene as the prototype. The CCSD(T)/CBS reference data show its strength amounting to -10.2 kcal mol(-1), comparable to a typical H-bond or an anion-π interaction. The energy decomposition analysis reveals that the aerogen-π interaction is favored by the electrostatic interaction (27.7%), the induction (13.4%), and the dispersion (21.6%). This interaction may prompt us to consider the noncovalent chemistry of aerogen derivatives in the near future. PMID:26282579

  7. Enhanced Aerogen-π Interaction by a Cation-π Force.

    PubMed

    Miao, Junjian; Song, Bo; Gao, Yi

    2016-02-18

    The interaction between a noble gas atom and an aromatic π-electron system, which mainly originates from the London dispersion force, is very weak and has not attracted enough attention yet. Herein, we reported a type of notably enhanced aerogen-π interaction between cation-π systems and noble gas atoms. The binding strength of a divalent cation-π system with a xenon atom is comparable to a moderate hydrogen bond (up to ca. 7 kcal mol(-1)), whereas krypton and argon atoms produce slightly weaker interactions. Energy-decomposition analysis reveals that the induction interaction is responsible for the stabilization of divalent cation-π⋅Xe species besides the dispersion interaction. Our results might be helpful to increase the understanding of some unsolved mysteries of aerogens. PMID:26699400

  8. Enhanced dark hydrogen fermentation by addition of ferric oxide nanoparticles using Enterobacter aerogenes.

    PubMed

    Lin, Richen; Cheng, Jun; Ding, Lingkan; Song, Wenlu; Liu, Min; Zhou, Junhu; Cen, Kefa

    2016-05-01

    Ferric oxide nanoparticles (FONPs) were used to facilitate dark hydrogen fermentation using Enterobacter aerogenes. The hydrogen yield of glucose increased from 164.5±2.29 to 192.4±1.14mL/g when FONPs concentration increased from 0 to 200mg/L. SEM images of E. aerogenes demonstrated the existence of bacterial nanowire among cells, suggesting FONPs served as electron conduits to enhance electron transfer. TEM showed cellular internalization of FONPs, indicating hydrogenase synthesis and activity was potentially promoted due to the released iron element. When further increasing FONPs concentration to 400mg/L, the hydrogen yield of glucose decreased to 147.2±2.54mL/g. Soluble metabolic products revealed FONPs enhanced acetate pathway of hydrogen production, but weakened ethanol pathway. This shift of metabolic pathways allowed more nicotinamide adenine dinucleotide for reducing proton to hydrogen. PMID:26890796

  9. Is Aerogen-π Interaction Capable of Initiating the Noncovalent Chemistry of Group 18?

    PubMed

    Miao, Junjian; Song, Bo; Gao, Yi

    2015-12-01

    The interactions between atoms of noble gases and π systems are generally considered as van der Waals interaction, which have not attracted attention yet. Herein, we present high-level ab initio calculations to show the unexpected noncovalent interaction between a covalently bonded noble gas atom and a delocalized aromatic π electron using XeO3⋅benzene as the prototype. The CCSD(T)/CBS reference data show its strength amounting to -10.2 kcal mol(-1), comparable to a typical H-bond or an anion-π interaction. The energy decomposition analysis reveals that the aerogen-π interaction is favored by the electrostatic interaction (27.7%), the induction (13.4%), and the dispersion (21.6%). This interaction may prompt us to consider the noncovalent chemistry of aerogen derivatives in the near future.

  10. Engineered Enterobacter aerogenes for efficient utilization of sugarcane molasses in 2,3-butanediol production.

    PubMed

    Jung, Moo-Young; Park, Bu-Soo; Lee, Jinwon; Oh, Min-Kyu

    2013-07-01

    Sugarcane molasses is considered to be a good carbon source for biorefinery due to its high sugar content and low price. Sucrose occupies more than half of the sugar in the molasses. Enterobacter aerogenes is a good host strain for 2,3-butanediol production, but its utilization of sucrose is not very efficient. To improve sucrose utilization in E. aerogenes, a sucrose regulator (ScrR) was disrupted from the genomic DNA. The deletion mutation increased the sucrose consumption rate significantly when sucrose or sugarcane molasses was used as a carbon source. The 2,3-butanediol production from sugarcane molasses by the mutant was enhanced by 60% in batch fermentation compared to that by the wild type strain. In fed-batch fermentation, 98.69 g/L of 2,3-butanediol production was achieved at 36 h.

  11. Purification and antibiofilm activity of AHL-lactonase from endophytic Enterobacter aerogenes VT66.

    PubMed

    Rajesh, P S; Rai, V Ravishankar

    2015-11-01

    The opportunistic pathogen Pseudomonas aeruginosa uses biofilm lifestyle to resist antibiotic treatment. In our study, endophytic bacterium Enterobacter aerogenes VT66 quenched the N-acyl homoserine lactone (AHL) molecules produced by P. aeruginosa PAO1. The quorum quenching activity was attributed to the presence of AHL-lactonase. The AHL-lactonase was purified using column chromatography and purified AHL-lactonase was applied for the control of biofilm formation in P. aeruginosa PAO1. The results showed that purified AHL-lactonase obtained with a molecular weight about 30kDa was able to inhibit more than 70% of biofilm in P. aeruginosa PAO1 (P<0.001). Antibiofilm activity of AHL-lactonase was correlated well with results from staining technique used to determine inhibition of biomass and viable cell activity. Therefore, results unambiguously confirm that the AHL-lactonase from E. aerogenes VT66 could be used as antibiofilm therapeutics in P. aeruginosa associated biomedical applications.

  12. Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates.

    PubMed

    Lavigne, J-P; Sotto, A; Nicolas-Chanoine, M-H; Bouziges, N; Bourg, G; Davin-Regli, A; Pagès, J-M

    2012-06-01

    Imipenem-susceptible E. aerogenes isolates exhibiting extended spectrum β-lactamases, target mutations and a basal efflux expression, were identified in five patients. After imipenem treatment, imipenem-intermediate susceptible (IMI-I) or resistant (IMI-R) isolates emerged in these patients. Alteration in porin synthesis and increase in efflux expression were observed in the IMI-I isolates whereas complete loss of the porins, LPS alteration and efflux overexpression were observed in the IMI-R isolates. Bacterial virulence of the strains was investigated by the Caenorhabditis elegans model. The IMI-R isolates were shown to be significantly less virulent than the IMI-susceptible or IMI-I isolates. The pleiotropic membrane alteration and its associated fitness burden exhibited by E. aerogenes isolates influence their antibiotic resistance and their virulence behaviour. These findings highlight the balance between the low permeability-related resistance and virulence and their relationships with the treatment of resistant pathogens.

  13. Delivery of TEM beta-lactamase by gene-transformed Lactococcus lactis subsp. lactis through cervical cell monolayer.

    PubMed

    Kaushal, Gagan; Trombetta, Louis; Ochs, Raymond S; Shao, Jun

    2006-04-26

    Lactococcus lactis subsp. lactis transformed with Plasmid ss80 (encoding the production and secretion of TEM beta-lactamase) was used for the delivery of beta-lactamase through the C-33A (cervix cell) monolayer. The viability of the cell monolayers co-cultured with L. lactis was examined by the trypan blue exclusion method. The integrity of the monolayers was monitored by measuring the transport of mannitol and propranolol as well as the transepithelial electrical resistance. The transport rate of beta-lactamase through C-33A monolayer was increased by four- and nine-folds (p < 0.05) at the first hour by the transformed L. lactis compared to the free solution with or without presence of the untransformed L. lactis, respectively. This increase was gradually diminished after the 1st hour: it became 30 and 50% (p < 0.05) at 10 h. The presence of the untransformed L. lactis with free solution delivery also increased the transport rate by 100% at 1 h (p < 0.05) and 15% at 10h (p>0.05). The increase in transport rate by the transformed L. lactis is most probably due to the concentrate of beta-lactamase on C-33A monolayer. When co-cultured with the L. lactis, the C-33A cell viability and the monolayer TEER remained steady for 10 h. The presence of L. lactis did not change the transport of propranolol and mannitol through the monolayers. In conclusion, the transformed L. lactis significantly (p < 0.05) increased the transport of beta-lactamase through the cervical monolayers, indicating probiotic bacteria delivery may be a promising approach for protein delivery through the vagina.

  14. Stability and activity of an Enterobacter aerogenes-specific bacteriophage under simulated gastro-intestinal conditions.

    PubMed

    Verthé, K; Possemiers, S; Boon, N; Vaneechoutte, M; Verstraete, W

    2004-09-01

    A bacteriophage, designated UZ1 and showing lytic activity against a clinically important strain (BE1) of Enterobacter aerogenes was isolated from hospital sewage. The stability and lytic activity against this strain under simulated gastro-intestinal conditions was evaluated. After addition of bacteriophage UZ1 to a liquid feed at gastric pH 2, the phage was immediately inactivated and could not be recovered. However, by use of an antacid to neutralize stomach acidity, no significant changes in phage titer were observed after 2 h incubation at 37 degrees C. After supplementing pancreatic juice and further incubation for 4 h, the phage titer remained stable. The persistence of UZ1 in a mixed microbial ecosystem that was representative for the large intestine was monitored using an in vitro simulation of the human intestinal microbial ecosystem. A pulse administration of bacteriophage UZ1 at a concentration of 10(5) plaque-forming units (PFU)/ml to reactor 3 (which simulates the ascending colon) showed that, in the absence of the host, bacteriophage UZ1 persisted for 13 days in the simulated colon, while the theoretical washout was calculated at 16 days. To assess its lytic activity in an intestinal microbial ecosystem, a green fluorescent protein (gfp)-labeled E. aerogenes BE1 strain was constructed and gfp-specific primers were designed in order to quantify the host strain using real-time PCR. It was observed that bacteriophage UZ1 was able to replicate and showed lytic activity against E. aerogenes BE1/ gfp in an intestinal microbial ecosystem. Indeed, after 17 h a 2 log unit reduction of E. aerogenes BE1/ gfp was measured as compared with the assay without bacteriophage UZ1, while the phage titer increased by 2 log units at an initial multiplicity of infection of 0.07 PFU/colony-forming unit. This is the first report of an in vitro model to study bacteriophage activity in the complex intestinal microbial community.

  15. Biotransformation of ferulic acid to 4-vinylguaiacol by Enterobacter soli and E. aerogenes.

    PubMed

    Hunter, William J; Manter, Daniel K; van der Lelie, Daniel

    2012-12-01

    We investigated the conversion of ferulic acid to 4-vinylguaiacol (4-VG), vanillin, vanillyl alcohol, and vanillic acid by five Enterobacter strains. These high-value chemicals are usually synthesized by chemical methods but biological synthesis adds market value. Ferulic acid, a relatively inexpensive component of agricultural crops, is plentiful in corn hulls, cereal bran, and sugar-beet pulp. Two Enterobacter strains, E. soli, and E. aerogenes, accumulated 550-600 ppm amounts of 4-VG when grown in media containing 1,000 ppm ferulic acid; no accumulations were observed with the other strains. Decreasing the amount of ferulic acid present in the media increased the conversion efficiency. When ferulic acid was supplied in 500, 250, or 125 ppm amounts E. aerogenes converted ~72 % of the ferulic acid present to 4-VG while E. soli converted ~100 % of the ferulic acid to 4-VG when supplied with 250 or 125 ppm amounts of ferulic acid. Also, lowering the pH improved the conversion efficiency. At pH 5.0 E. aerogenes converted ~84 % and E. soli converted ~100 % of 1,000 ppm ferulic acid to 4-VG. Only small, 1-5 ppm, accumulations of vanillin, vanillyl alcohol, and vanillic acid were observed. E. soli has a putative phenolic acid decarboxylase (PAD) that is 168 amino acids long and is similar to PADs in other enterobacteriales; this protein is likely involved in the bioconversion of ferulic acid to 4-VG. E. soli or E. aerogenes might be useful as a means of biotransforming ferulic acid to 4-VG.

  16. Biodegradation of 2-methylquinoline by Enterobacter aerogenes TJ-D isolated from activated sludge.

    PubMed

    Wang, Lin; Li, Yongmei; Duan, Jingyuan

    2013-07-01

    Bacterial strain Enterobacter aerogenes TJ-D capable of utilizing 2-methylquinoline as the sole carbon and energy source was isolated from acclimated activated sludge under denitrifying conditions. The ability to degrade 2-methylquinoline by E. aerogenes TJ-D was investigated under denitrifying conditions. Under optimal conditions of temperature (35 degrees C) and initial pH 7, 2-methylquinoline of 100 mg/L was degraded within 176 hr. The degradation of 2-methylquinoline by E. aerogenes TJ-D could be well described by the Haldane model (R2 > 0.91). During the degradation period of 2-methylquinoline (initial concentration 100 mg/L), nitrate was almost completely consumed (the removal efficiency was 98.5%), while nitrite remained at low concentration (< 0.62 mg/L) during the whole denitrification period. 1,2,3,4-Tetrahydro-2-methylquinoline, 4-ethyl-benzenamine, N-butyl-benzenamine, N-ethyl-benzenamine and 2,6-diethyl-benzenamine were metabolites produced during the degradation. The degradation pathway of 2-methylquinoline by E. aerogenes TJ-D was proposed. 2-Methylquinoline is initially hydroxylated at C-4 to form 2-methyl-4-hydroxy-quinoline, and then forms 2-methyl-4-quinolinol as a result of tautomerism. Hydrogenation of the heterocyclic ring at positions 2 and 3 produces 2,3-dihydro-2-methyl-4-quinolinol. The carbon-carbon bond at position 2 and 3 in the heterocyclic ring may cleave and form 2-ethyl-N-ethyl-benzenamine. Tautomerism may result in the formation of 2,6-diethyl-benzenamine and N-butyl-benzenamine. 4-Ethyl-benzenamine and N-ethyl-benzenamine were produced as a result of losing one ethyl group from the above molecules.

  17. Robust Multivariable Flutter Suppression for the Benchmark Active Control Technology (BACT) Wind-Tunnel Model

    NASA Technical Reports Server (NTRS)

    Waszak, Martin R.

    1997-01-01

    The Benchmark Active Controls Technology (BACT) project is part of NASA Langley Research Center s Benchmark Models Program for studying transonic aeroelastic phenomena. In January of 1996 the BACT wind-tunnel model was used to successfully demonstrate the application of robust multivariable control design methods (H and -synthesis) to flutter suppression. This paper addresses the design and experimental evaluation of robust multivariable flutter suppression control laws with particular attention paid to the degree to which stability and performance robustness was achieved.

  18. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment

    PubMed Central

    Davin-Regli, Anne; Pagès, Jean-Marie

    2015-01-01

    Enterobacter aerogenes and E. cloacae have been reported as important opportunistic and multiresistant bacterial pathogens for humans during the last three decades in hospital wards. These Gram-negative bacteria have been largely described during several outbreaks of hospital-acquired infections in Europe and particularly in France. The dissemination of Enterobacter sp. is associated with the presence of redundant regulatory cascades that efficiently control the membrane permeability ensuring the bacterial protection and the expression of detoxifying enzymes involved in antibiotic degradation/inactivation. In addition, these bacterial species are able to acquire numerous genetic mobile elements that strongly contribute to antibiotic resistance. Moreover, this particular fitness help them to colonize several environments and hosts and rapidly and efficiently adapt their metabolism and physiology to external conditions and environmental stresses. Enterobacter is a versatile bacterium able to promptly respond to the antibiotic treatment in the colonized patient. The balance of the prevalence, E. aerogenes versus E. cloacae, in the reported hospital infections during the last period, questions about the horizontal transmission of mobile elements containing antibiotic resistance genes, e.g., the efficacy of the exchange of resistance genes Klebsiella pneumoniae to Enterobacter sp. It is also important to mention the possible role of antibiotic use in the treatment of bacterial infectious diseases in this E. aerogenes/E. cloacae evolution. PMID:26042091

  19. Most Enterobacter aerogenes Strains in France Belong to a Prevalent Clone

    PubMed Central

    Bosi, Claude; Davin-Regli, Anne; Bornet, Charleric; Mallea, Monique; Pages, Jean-Marie; Bollet, Claude

    1999-01-01

    The aim of this study was to determine the distribution in France of the Enterobacter aerogenes prevalent clone isolated in the hospitals of the Marseille area (A. Davin-Regli, D. Monnet, P. Saux, C. Bosi, R. Charrel, A. Barthelemy, and C. Bollet, J. Clin. Microbiol. 34:1474–1480, 1996). A total of 123 E. aerogenes isolates were collected from 23 hospital laboratories and analyzed by random amplification of polymorphic DNA and enterobacterial repetitive intergenic consensus-PCR to determine their epidemiological relatedness. Molecular typing revealed that 21 of the 23 laboratories had isolated this prevalent clone harboring the plasmid encoding for extended-spectrum β-lactamase of the TEM-24 type. Most isolates were susceptible only to imipenem and gentamicin. Their dissemination seems to be clonal and was probably the result of the general use of broad-spectrum cephalosporins and quinolones. Four isolates showed an alteration of their outer membrane proteins, causing decrease of susceptibility to third-generation cephalosporins and imipenem and leading to the critical situation of having no alternative therapeutic. The large dissemination of the E. aerogenes prevalent clone probably results from its good adaptation to the antibiotics administered in France and the hospital environment, particularly in intensive care units. PMID:10364580

  20. Partial purification and characterization of a novel histidine decarboxylase from Enterobacter aerogenes DL-1.

    PubMed

    Zou, Yu; Hu, Wenzhong; Jiang, Aili; Tian, Mixia

    2015-08-18

    Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca(2+) and Mn(2+) showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver-Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

  1. Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production.

    PubMed

    Jung, Moo-Young; Ng, Chiam Yu; Song, Hyohak; Lee, Jinwon; Oh, Min-Kyu

    2012-07-01

    2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

  2. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment.

    PubMed

    Davin-Regli, Anne; Pagès, Jean-Marie

    2015-01-01

    Enterobacter aerogenes and E. cloacae have been reported as important opportunistic and multiresistant bacterial pathogens for humans during the last three decades in hospital wards. These Gram-negative bacteria have been largely described during several outbreaks of hospital-acquired infections in Europe and particularly in France. The dissemination of Enterobacter sp. is associated with the presence of redundant regulatory cascades that efficiently control the membrane permeability ensuring the bacterial protection and the expression of detoxifying enzymes involved in antibiotic degradation/inactivation. In addition, these bacterial species are able to acquire numerous genetic mobile elements that strongly contribute to antibiotic resistance. Moreover, this particular fitness help them to colonize several environments and hosts and rapidly and efficiently adapt their metabolism and physiology to external conditions and environmental stresses. Enterobacter is a versatile bacterium able to promptly respond to the antibiotic treatment in the colonized patient. The balance of the prevalence, E. aerogenes versus E. cloacae, in the reported hospital infections during the last period, questions about the horizontal transmission of mobile elements containing antibiotic resistance genes, e.g., the efficacy of the exchange of resistance genes Klebsiella pneumoniae to Enterobacter sp. It is also important to mention the possible role of antibiotic use in the treatment of bacterial infectious diseases in this E. aerogenes/E. cloacae evolution.

  3. Inactivation of Enterobacter aerogenes in reconstituted skim milk by high- and low-frequency ultrasound.

    PubMed

    Gao, Shengpu; Hemar, Yacine; Lewis, Gillian D; Ashokkumar, Muthupandian

    2014-11-01

    The inactivation of Enterobacter aerogenes in skim milk using low-frequency (20kHz) and high-frequency (850kHz) ultrasonication was investigated. It was found that low-frequency acoustic cavitation resulted in lethal damage to E. aerogenes. The bacteria were more sensitive to ultrasound in water than in reconstituted skim milk having different protein concentrations. However, high-frequency ultrasound was not able to inactivate E. aerogenes in milk even when powers as high as 50W for 60min were used. This study also showed that high-frequency ultrasonication had no influence on the viscosity and particle size of skim milk, whereas low-frequency ultrasonication resulted in the decrease in viscosity and particle size of milk. The decrease in particle size is believed to be due to the breakup of the fat globules, and possibly to the cleavage of the κ-casein present at the surface of the casein micelles. Whey proteins were also found to be slightly affected by low-frequency ultrasound, with the amounts of α-lactalbumin and β-lactoglobulin slightly decreasing.

  4. Regulation of glycerol metabolism in Enterobacter aerogenes NBRC12010 under electrochemical conditions.

    PubMed

    Hatayama, Kouta; Yagishita, Tatsuo

    2009-06-01

    Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose. These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed reaction mainly depended on the ratio of NAD(+)/NADH. At a NAD(+)/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance of cellular NAD(+)/NADH. Exogenous NADH was oxidized to NAD(+) by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical conditions.

  5. Characteristics of the melibiose transporter and its primary structure in Enterobacter aerogenes.

    PubMed

    Okazaki, N; Kuroda, M; Shimamoto, T; Shimamoto, T; Tsuchiya, T

    1997-05-22

    Cells of Enterobacter aerogenes can grow on melibiose as a sole source of carbon. This suggests the presence of melibiose operon in this organism. We found that E. aerogenes cells possess both alpha-galactosidase activity and melibiose transport activity, which were induced by melibiose. Neither Na+ nor Li+ stimulated the melibiose transport. However, transport of methyl-beta-thiogalactoside (TMG) was stimulated by Li+ but not by Na+. These findings suggest that the major coupling cation for the melibiose transporter in E. aerogenes is H+. In fact, we observed H+ entry into cells caused by an influx of melibiose and some of its analogs. We cloned the melB gene which encodes the melibiose transporter, and sequenced it. Deduced amino acid sequence of the transporter revealed that the melibiose transporter consists of 471 amino acid residues and the molecular weight was calculated to be 52214 Da. The sequence showed high homology with the sequences of the melibiose transporters of Escherichia coli, Salmonella typhimurium and Klebsiella pneumoniae. Higher homology was found with the melibiose transporter of K. pneumoniae than with that of E. coli and S. typhimurium.

  6. Anthranilate synthase/anthranilate 5-phosphoribosyl 1-pyrophosphate phosphoribosyltransferase from Aerobacter aerogenes

    PubMed Central

    Egan, A. F.; Gibson, F.

    1972-01-01

    1. Anthranilate synthase and phosphoribosyltransferase from Aerobacter aerogenes purify simultaneously and sediment together on sucrose gradients, showing that they occur as an enzyme aggregate. Both activities of the intact aggregate are subject to inhibition by tryptophan. 2. By using appropriate auxotrophic mutants it was shown that an intact active enzyme aggregate is formed when the components come from separate mutant strains. An intact active aggregate can also be formed when one component is from Escherichia coli and the other from A. aerogenes. 3. Phosphoribosyltransferase of A. aerogenes is active when not in an aggregate with anthranilate synthase, but is not subject to tryptophan inhibition, indicating that the inhibitor site is on the anthranilate synthase component. 4. Anthranilate synthase can be active and sensitive to tryptophan inhibition when complexed with an inactive phosphoribosyltransferase. 5. Kinetic studies on the anthranilate synthase activity show that tryptophan is a competitive inhibitor with respect to chorismate and a non-competitive inhibitor with respect to either glutamine or NH4+ ions. This is consistent with a sequential mechanism of the ordered type in which chorismate is the first reactant. PMID:4352716

  7. Occurrence of efflux mechanism and cephalosporinase variant in a population of Enterobacter aerogenes and Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases.

    PubMed

    Tran, Que-Tien; Dupont, Myrielle; Lavigne, Jean-Philippe; Chevalier, Jacqueline; Pagès, Jean-Marie; Sotto, Albert; Davin-Regli, Anne

    2009-04-01

    We investigated the occurrence of multidrug resistance in 44 Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Efflux was involved in resistance in E. aerogenes isolates more frequently than in K. pneumoniae isolates (100 versus 38% of isolates) and was associated with the expression of phenylalanine arginine beta-naphthylamide-susceptible active efflux. AcrA-TolC overproduction in E. aerogenes isolates was noted. An analysis of four E. aerogenes isolates for which cefepime MICs were high revealed no modification in porin expression but a new specific mutation in the AmpC beta-lactamase.

  8. Ornithine transport and exchange in Streptococcus lactis

    SciTech Connect

    Thompson, J.

    1987-09-01

    Resting cells of Streptococcus lactis 133 appeared to accumulate (/sup 14/C)ornithine to a high concentration in the absence of an exogenous energy source. However, analysis of intracellular amino acid pool constituents and results of transport experiments revealed that the accumulation of ornithine represented a homoexchange between extracellular (/sup 14/C)ornithine and unlabeled ornithine in the cell. The energy-independent exchange of ornithine was not inhibited by proton-conducting uncouplers or by metabolic inhibitors. Intracellular (/sup 14/C)ornithine was retained by resting cells after suspension in a buffered medium. However, addition of unlabeled ornithine to the suspension elicited rapid exit of labeled amino acid. The initial rate of exist of (/sup 14/C)ornithine was dependent on the concentration of unlabeled ornithine in the medium, but this accelerative exchange diffusion process caused no net loss of amino acid. By contrast, the presence of a fermentable energy source caused a rapid expulsion of and new decrease in the concentration of intracellular ornithine. Kinetic analyses of amino acid transport demonstrated competitive inhibition between lysine and ornithine, and data obtained by two-dimensional thin-layer chromatography established the heteroexchange of these basic amino acids. The effects of amino acids and of ornithine analogs on both entry and exit of (/sup 14/C)ornithine have been examined. The data suggest that common carrier mediates the entry and exchange of lysine, arginine, and ornithine in cells of S. lactis.

  9. Membrane protein expression in Lactococcus lactis.

    PubMed

    King, Martin S; Boes, Christoph; Kunji, Edmund R S

    2015-01-01

    The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels. PMID:25857778

  10. Identification and characteristics of nisin Z-producing Lactococcus lactis subsp. lactis isolated from Kimchi.

    PubMed

    Park, Sang-Hee; Itoh, Kikuji; Kikuchi, Eisaku; Niwa, Hidekazu; Fujisawa, Tomohiko

    2003-05-01

    We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100 degrees C for 1 h and at 121 degrees C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or beta-glucosidase. There were some differences in characteristics from those of nisins described previously. PMID:12732968

  11. Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media.

    PubMed

    McIntyre, D A; Harlander, S K

    1989-10-01

    The impact of growth conditions on electroporation of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) was evaluated. Cells grown in M17 broth supplemented with 0.5% glucose (M17-Glu) and two chemically defined synthetic media, FMC and RPMI 1640, all supplemented with 0.24% DL-threonine or 0.5% glycine, were harvested, washed with double-distilled water, diluted, and porated in the presence of 1 microgram of pGB301 DNA with a Transfector 100 (BTX, Inc., San Diego, Calif.) or a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.). Transformants were recovered at consistently higher efficiencies for cells grown in FMC or RPMI 1640 (10(3) to 10(4) transformants per micrograms of DNA) than for cells grown in M17-Glu (10(1) to 10(2) transformants per micrograms of DNA). Other parameters influencing electroporation of L. lactis cells grown in chemically defined media were growth phase and final concentration of cells, concentration of plasmid DNA, voltage achieved during poration, and expression conditions. A high degree of variability in transformation efficiencies was evident for replicate samples of cells pulsed with either electroporation machine. A trend toward decreased variability was observed for duplicate samples of cells prepared on the same day. In addition, storage studies done with a large batch of cells prepared on the same day indicated that freezing dry cell pellets at -60 degrees C had no deleterious effect on transformation efficiencies over a 30-day period when a new 0.2-cm cuvette was used for porating each sample. PMID:2513778

  12. Membrane H+ conductance of Streptococcus lactis.

    PubMed Central

    Maloney, P C

    1979-01-01

    Membrane conductance to H+ was measured in the anaerobic bacterium Streptoccus lactis by a pulse technique employing a low driving force (0.1 pH unit; 6 mV). Over the pH range of 3.7 to 8.5, a constant value for passive H+ conductance was observed, corresponding to 0.2 mumol of H+/s per p/ unit per g, dry weight (1.6 microS/cm2 of surface area). The pH insensitivity of this low basal H+ conductance supports the idea that a circulation of protons can mediate highly efficiency engery transductions across the membranes of bacteria. PMID:40951

  13. [Application of gas chromatography in the identification of Enterobacter cloacae, Enterobacter aerogenes, and Enterobacter agglomerans].

    PubMed

    Robles Valderrama, E; Ramírez García, P; González Arreaga, M E; Sáinz Morales, M G; Martínez Rodríguez, B; Durán Díaz, A; Chávez Ramírez, D

    1999-01-01

    Enterobacter cloacae, Enterobacter aerogenes and Enterobacter agglomerans were identified using gas chromatography as a substitution of the traditional techniques. Their acid methyl esters profiles were determined using a gas chromatograph Hewlett Packard 5890A and a RSL-150 heliflex capillary column. A total of 120 samples were analyzed from reference strains (ATCC 13047, 13048, 27155) and environmental isolations, eleven fatty acids were included in the profiles from which cis-9, 10-methyleneoctadecanoic acid (peak 24), cis-9-hexadecenoic acid (peak 14), octadecanoic acid (peak 23) and dodecanoic acid (peak 3), were the most important for the differentiation of the three species analyzed.

  14. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme...

  15. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme...

  16. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme...

  17. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... prepared from yeast that has been grown in a pure culture fermentation and by using materials that are..., nontoxicogenic yeast Kluyveromyces lactis (previously named Saccharomyces lactis). It contains the enzyme...

  18. Genome Sequence of a Lactococcus lactis Strain Isolated from Salmonid Intestinal Microbiota.

    PubMed

    Opazo, Rafael; Gajardo, Felipe; Ruiz, Mauricio; Romero, Jaime

    2016-08-25

    Lactococcus lactis is a common inhabitant of the intestinal microbiota of salmonids, especially those in aquaculture systems. Here, we present a genome sequence of a Lactococcus lactis strain isolated from the intestinal contents of rainbow trout reared in Chile.

  19. Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis

    PubMed Central

    Bessonov, Sergey; Anokhina, Maria; Krasauskas, Andrius; Golas, Monika M.; Sander, Bjoern; Will, Cindy L.; Urlaub, Henning; Stark, Holger; Lührmann, Reinhard

    2010-01-01

    To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3′ splice site and 3′ exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human Bact complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to Bact and Bact to C transitions, and comparisons with the Saccharomyces cerevisiae Bact complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to Bact and Bact to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human Bact complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae Bact complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved. PMID:20980672

  20. Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

    PubMed

    Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes. PMID:27343449

  1. Isolation and characterization of a bacteriophage F20 virulent to Enterobacter aerogenes.

    PubMed

    Mishra, Charitra Kumar; Choi, Tae Jin; Kang, Sun Chul

    2012-10-01

    An aquatic phage, designated F20, was characterized and its physico-chemical characteristics studied. F20 was specifically virulent to only two strains of Enterobacter aerogenes (ATCC 13048 and the multi-drug-resistant strain K113) among other species tested (n = 15). It was classified in the family Siphoviridae of T1-like viruses and contained a linear dsDNA genome estimated to be 51.5 kbp enclosed by an isometric capsid of 50±2 nm in diameter and a tail of 150±3 nm in length. F20 was able to survive in a broad pH range between 4 and 11, showed potential for future animal trials using oral solution and resisted chloroform and ethanol. It exhibited remarkable stability between room temperature and 70 °C for up to 150 min, and even up to 6 months at room temperature. Knowledge of this phage belonging to the widespread T1-like viruses might be helpful for adopting therapeutic strategies against E. aerogenes.

  2. Hospital clonal dissemination of Enterobacter aerogenes producing carbapenemase KPC-2 in a Chinese teaching hospital.

    PubMed

    Qin, Xiaohua; Yang, Yang; Hu, Fupin; Zhu, Demei

    2014-02-01

    Carbapenems are first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant Enterobacteriaceae. However, resistance to carbapenems has increased dramatically among Enterobacteriaceae in our hospital. In this study, we report clonal dissemination caused by carbapenem-resistant Enterobacter aerogenes (CREA). In 2011, CREA was identified from 12 patients admitted to the neurosurgical ward. All 12 clinical isolates were non-susceptible to cefotaxime, ceftazidime, cefoxitin, ertapenem, imipenem or meropenem. All isolates carried the gene encoding Klebsiella pneumoniae carbapenemase-2 (KPC-2), except for the isolate E4. However, a remarkably lower expression level of the porin OmpF was detected in the non-KPC-2-producing isolate E4 on SDS-PAGE compared with the carbapenem-susceptible isolate. Epidemiological and molecular investigations showed that a single E. aerogenes strain (PFGE type A), including seven KPC-2-producing clinical isolates, was primarily responsible for the first isolation and subsequent dissemination. In a case-control study, we identified risk factors for infection/colonization with CREA. Mechanical ventilation, the changing of sickbeds and previous use of broad-spectrum antibiotics were identified as potential risk factors. Our findings suggest that further studies should focus on judicious use of available antibiotics, implementation of active antibiotic resistance surveillance and strict implementation of infection-control measures to avoid the rapid spread or clonal dissemination caused by carbapenem-resistant Enterobacteriaceae in healthcare facilities.

  3. [Isolation and identification of degradation bacteria Enterobacter aerogenes for pyrethriods pesticide residues and its degradation characteristics].

    PubMed

    Liao, Min; Zhang, Hai-jun; Xie, Xiao-mei

    2009-08-15

    By incubation experiment, the bacterial strain labeled as M6R9 was isolated from the tame sludge in water course of Pesticide Factory of Hangzhou, and was identified as Enterobacter aerogenes, which had highly efficient degradation for Bifenthrin, Fenpropathrin and Cypermethrin. By investigating the physiological characteristics of the strain, the results show that the bacterium is a gram-negative aerobe bacilli, size is (0.8-1.9) microm x (0.5-1.0) microm, and is capable of utilizing Bifenthrin, Fenpropathrin and Cypermethrin as sole carbon source. Under the condition of ventilation, (25-30) degrees C, inoculated amount at D(415 nm) 0.2, pH 7.0, pesticide concentration 100 mg x L(-1) and vibrational speed 180 r x min(-1), the degradation efficiencies to Bifenthrin, Fenpropathrin and Cypermethrin are the highest by strain M6R9. Under such condition, in the mixture culture medium with 100 mg x L(-1) Bifenthrin, Fenpropathrin and Cypermethrin, the degradation ratios are 55.74%, 55.11% and 55.96% after culturing 3 d, respectively, the degradation processes are fitted for first-order kinetic equation and the half lives (t(1/2)) are 65.4,70.7 and 68.6 h respectively. The degradation ability of Enterobacter aerogenes M6R9 on Bifenthrin, Fenpropathrin and Cypermethrin is positively correlated to inoculated amount,vibrational speed and ventilation.

  4. Enhancing hydrogen production of Enterobacter aerogenes by heterologous expression of hydrogenase genes originated from Synechocystis sp.

    PubMed

    Song, Wenlu; Cheng, Jun; Zhao, Jinfang; Zhang, Chuanxi; Zhou, Junhu; Cen, Kefa

    2016-09-01

    The hydrogenase genes (hoxEFUYH) of Synechocystis sp. PCC 6803 were cloned and heterologously expressed in Enterobacter aerogenes ATCC13408 for the first time in this study, and the hydrogen yield was significantly enhanced using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into E. aerogenes ATCC13408 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the hox genes. The hydrogenase activity of the recombinant strain is 237.6±9.3ml/(g-DW·h), which is 152% higher than the wild strain. The hydrogen yield of the recombinant strain is 298.3ml/g-glucose, which is 88% higher than the wild strain. During hydrogen fermentation, the recombinant strain produces more acetate and butyrate, but less ethanol. This is corresponding to the NADH metabolism in the cell due to the higher hydrogenase activity with the heterologous expression of hox genes.

  5. Effect of nanocomposite packaging containing ZnO on growth of Bacillus subtilis and Enterobacter aerogenes.

    PubMed

    Esmailzadeh, Hakimeh; Sangpour, Parvaneh; Shahraz, Farzaneh; Hejazi, Jalal; Khaksar, Ramin

    2016-01-01

    Recent advances in nanotechnology have opened new windows in active food packaging. Nano-sized ZnO is an inexpensive material with potential antimicrobial properties. The aim of the present study is to evaluate the antibacterial effect of low density Polyethylene (LDPE) containing ZnO nanoparticles on Bacillus subtilis and Enterobacter aerogenes. ZnO nanoparticles have been synthesized by facil molten salt method and have been characterized by X-ray diffraction (XRD), and scanning electron microscopy (SEM). Nanocomposite films containing 2 and 4 wt.% ZnO nanoparticles were prepared by melt mixing in a twin-screw extruder. The growth of both microorganisms has decreased in the presence of ZnO containing nanocomposites compared with controls. Nanocomposites with 4 wt.% ZnO nanoparticles had stronger antibacterial effect against both bacteria in comparison with the 2 wt.% ZnO containing nanocomposites. B. subtilis as Gram-positive bacteria were more sensitive to ZnO containing nanocomposite films compared with E. aerogenes as Gram-negative bacteria. There were no significant differences between the migration of Zn ions from 2 and 4 wt.% ZnO containing nanocomposites and the released Zn ions were not significantly increased in both groups after 14 days compared with the first. Regarding the considerable antibacterial effects of ZnO nanoparticles, their application in active food packaging can be a suitable solution for extending the shelf life of food.

  6. The purification, crystallization and preliminary diffraction of a glycerophosphodiesterase from Enterobacter aerogenes

    SciTech Connect

    Jackson, Colin J.; Carr, Paul D.; Kim, Hye-Kyung; Liu, Jian-Wei; Ollis, David L.

    2006-07-01

    The metallo-glycerophosphodiesterase from E. aerogenes (GpdQ) has been cloned, expressed in E. coli and purified. Initial screening of crystallization conditions for this enzyme resulted in the identification of needles from one condition in a sodium malonate grid screen. Removal of the metals from the enzyme and subsequent optimization of these conditions led to crystals. The metallo-glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) has been cloned, expressed in Escherichia coli and purified. Initial screening of crystallization conditions for this enzyme resulted in the identification of needles from one condition in a sodium malonate grid screen. Removal of the metals from the enzyme and subsequent optimization of these conditions led to crystals that diffracted to 2.9 Å and belonged to space group P2{sub 1}3, with unit-cell parameter a = 164.1 Å. Self-rotation function analysis and V{sub M} calculations indicated that the asymmetric unit contains two copies of the monomeric enzyme, corresponding to a solvent content of 79%. It is intended to determine the structure of this protein utilizing SAD phasing from transition metals or molecular replacement.

  7. Effect of Some nitrosative agents on the growth of vgb-bearing Enterobacter aerogenes strains.

    PubMed

    Khleifat, Khaled M; Al-Mustafa, Ahmed H

    2007-07-01

    The effect of transnitrosation intermediate between S-nitroso-N-acetylcysteine (NACysNO) and cysteine on the growth of vgb-bearing Enterobacter aerogenes was investigated using three parameters: the ratio of the specific growth rates, the inhibition zone, and alpha-amylase synthesis for the culture exposed to stressors to that of the same stressor-free cultures. The effect of NACysNO/cysteine on the growth of Enterobacter strains was distinctive as compared with the CysNO, NACysNO, and their combination. At a higher concentration (2 mM), the extents of inhibition based on the mu(NACysNO/cysteine)/mu(no stress) ratio for these cultures were 57%, 62%, and 68% for VHb-expressing, parental, and pUC9-harboring cells, respectively. The inhibition caused by 2 mM: NACysNO in the presence of 1 mM cysteine in all bacterial strains was almost twofold that achieved by NACysNO alone. Based on the diameter of the inhibition zone and alpha-amylase productivity, the four compounds (NACysNO/Cysteine, CysNO, NACysNO, and their combinations) affected the E. aerogenes strains in a concentration-dependent and negative manner. This negative effect was lower in vgb-bearing than vgb-lacking strains. Thus, sulfur-to-sulfur transnitrosation was an efficient NO release and significantly (P < 0.05) affects the growth of Enterobacter strains, to a lesser extent in vgb-bearing strains.

  8. Study of the role of anaerobic metabolism in succinate production by Enterobacter aerogenes.

    PubMed

    Tajima, Yoshinori; Kaida, Kenichi; Hayakawa, Atsushi; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Fudou, Ryosuke; Matsui, Kazuhiko; Usuda, Yoshihiro; Sode, Koji

    2014-09-01

    Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain ΔadhE/PCK. The strain produced succinate from glucose with a 60.5% yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions.

  9. Purification and antibiofilm activity of AHL-lactonase from endophytic Enterobacter aerogenes VT66.

    PubMed

    Rajesh, P S; Rai, V Ravishankar

    2015-11-01

    The opportunistic pathogen Pseudomonas aeruginosa uses biofilm lifestyle to resist antibiotic treatment. In our study, endophytic bacterium Enterobacter aerogenes VT66 quenched the N-acyl homoserine lactone (AHL) molecules produced by P. aeruginosa PAO1. The quorum quenching activity was attributed to the presence of AHL-lactonase. The AHL-lactonase was purified using column chromatography and purified AHL-lactonase was applied for the control of biofilm formation in P. aeruginosa PAO1. The results showed that purified AHL-lactonase obtained with a molecular weight about 30kDa was able to inhibit more than 70% of biofilm in P. aeruginosa PAO1 (P<0.001). Antibiofilm activity of AHL-lactonase was correlated well with results from staining technique used to determine inhibition of biomass and viable cell activity. Therefore, results unambiguously confirm that the AHL-lactonase from E. aerogenes VT66 could be used as antibiofilm therapeutics in P. aeruginosa associated biomedical applications. PMID:26432367

  10. Impact of osmotic stress on protein diffusion in Lactococcus lactis.

    PubMed

    Mika, Jacek T; Schavemaker, Paul E; Krasnikov, Victor; Poolman, Bert

    2014-11-01

    We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram-positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ∼ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria.

  11. Large chromosomal inversion correlated with spectinomycin resistance in Lactococcus lactis subsp. lactis bv. diacetylactis S50.

    PubMed

    Kojic, Milan; Jovcic, Branko; Begovic, Jelena; Fira, Djordje; Topisirovic, Ljubisa

    2008-02-01

    A large chromosomal inversion that confers resistance to high concentrations of the antibiotic spectinomycin in Lactococcus lactis subsp. lactis bv. diacetylactis S50 was identified by pulsed field gel electrophoresis. The same type of inversion was identified in 4 independent experiments and in 4 different derivatives of strain S50, indicating the same position and the same mechanism of recombination as a response to antibiotic selective pressure in all derivatives. An analysis of ribosomal operons in strain S50 and mutants revealed that ribosomal operons are not endpoints of the recombination. Spectinomycin-resistant mutants appeared in a population of S50 derivatives at a high frequency of 2 x 10(-7). These spectinomycin-resistant mutants were not able to compete successfully with the wild-type strain during 25 generations (48 h) of co-culture in vitro, indicating that inversion had a significant fitness cost. Results demonstrate that as a mechanism of genome plasticity, inversion can be directly involved in one-step development of the adaptation to a high concentration of spectinomycin.

  12. The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp. lactis IL1403

    PubMed Central

    Bolotin, Alexander; Wincker, Patrick; Mauger, Stéphane; Jaillon, Olivier; Malarme, Karine; Weissenbach, Jean; Ehrlich, S. Dusko; Sorokin, Alexei

    2001-01-01

    Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group. [The sequence data described in this paper has been submitted to the GenBank data library under accession no. AE005176.] PMID:11337471

  13. Detecting the form of selection in the outer membrane protein C of Enterobacter aerogenes strains and Salmonella species.

    PubMed

    Padhi, Abinash; Verghese, Bindhu; Otta, Subhendu K

    2009-01-01

    The types of selective pressure operating on the outer membrane protein C (ompC) of Enterobacter aerogenes strains, the causative agent for nosocomial infections, and Salmonella sp., the hazardous pathogen are investigated using the maximum likelihood-based codon substitution models. Although the rate of amino acid replacement to the silent substitution (omega) across the entire codon sites of ompC of E. aerogenes (omega=0.3194) and Salmonella sp. (omega=0.2047) indicate that the gene is subjected to purifying selection (i.e. omega<1), approximately 3.7% of ompC codon sites in E. aerogenes (omega=21.52) are under the influence of positive Darwinian selection (i.e. omega>1). Such contrast in the intensity of selective pressures in both pathogens could be associated with the differential response to the adverse environmental changes. In E. aerogenes, majority of the positively selected sites are located in the hypervariable cell-surface-exposed domains whereas the trans-membrane domains are functionally highly constrained.

  14. Characterization of a cadmium resistance Lactococcus lactis subsp. lactis strain by antioxidant assays and proteome profiles methods.

    PubMed

    Sheng, Yao; Yang, Xuan; Lian, Yuanyuan; Zhang, Boyang; He, Xiaoyun; Xu, Wentao; Huang, Kunlun

    2016-09-01

    Heavy metal contamination poses a major threat to the environment and human health for their potential toxicity and non-biodegradable properties. At present, some probiotics bacteria are reported to have great potential to eliminate heavy metals from food and water. In this study, resistance properties of a newly isolated Lactococcus lactis subsp. lactis for cadmium were studied by antioxidant assays and proteomics analysis. Antioxidant capacity of this strain was significantly activated under cadmium stress indicated by Fenton reaction, DPPH assay, SOD assay and GSH assay. Intracellular antioxidant enzyme systems, such as superoxide dismutase, glutathione reductase and catalase were suggested to play vital roles in the activated antioxidant capacity. The up-regulated cadA was associated with the activated P-type ATPases that plays an important role in cadmium resistance. Proteomics analysis identified 12 over-expressed proteins under 50mg/L cadmium stress and these proteins are abundant in oxidative stress response and energy metabolism regulation, which were considered as consequences as cadmium resistance of the strain. Thus, the probiotics Lactococcus lactis subsp. lactis may resist cadmium stress through antioxidant approach and enhanced energy metabolism. The food grade lactis strain may be applied in metal decontamination in environment and food/feed. PMID:27522548

  15. Characterization of a cadmium resistance Lactococcus lactis subsp. lactis strain by antioxidant assays and proteome profiles methods.

    PubMed

    Sheng, Yao; Yang, Xuan; Lian, Yuanyuan; Zhang, Boyang; He, Xiaoyun; Xu, Wentao; Huang, Kunlun

    2016-09-01

    Heavy metal contamination poses a major threat to the environment and human health for their potential toxicity and non-biodegradable properties. At present, some probiotics bacteria are reported to have great potential to eliminate heavy metals from food and water. In this study, resistance properties of a newly isolated Lactococcus lactis subsp. lactis for cadmium were studied by antioxidant assays and proteomics analysis. Antioxidant capacity of this strain was significantly activated under cadmium stress indicated by Fenton reaction, DPPH assay, SOD assay and GSH assay. Intracellular antioxidant enzyme systems, such as superoxide dismutase, glutathione reductase and catalase were suggested to play vital roles in the activated antioxidant capacity. The up-regulated cadA was associated with the activated P-type ATPases that plays an important role in cadmium resistance. Proteomics analysis identified 12 over-expressed proteins under 50mg/L cadmium stress and these proteins are abundant in oxidative stress response and energy metabolism regulation, which were considered as consequences as cadmium resistance of the strain. Thus, the probiotics Lactococcus lactis subsp. lactis may resist cadmium stress through antioxidant approach and enhanced energy metabolism. The food grade lactis strain may be applied in metal decontamination in environment and food/feed.

  16. Acanthamoeba castellanii of the T4 genotype is a potential environmental host for Enterobacter aerogenes and Aeromonas hydrophila

    PubMed Central

    2013-01-01

    Background Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts. Methods Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with and invasion of A. castellanii. Additionally, bacterial survival intracellular of A. castellanii trophozoites as well as cysts was determined. Results All three bacterial isolates tested, associated, invaded, and survived inside A. castellanii trophozoites as well as A. castellanii cysts. However, E. aerogenes and E. coli exhibited significantly reduced association with and invasion of A. castellanii as compared with A. hydrophila (P < 0.01 using paired T-test, one tail distribution). In the long term survival assays, all three bacterial isolates tested remained viable inside A. castellanii trophozoites, while amoeba remained intact; however A. hydrophila exhibited higher survival inside amoebae (14.54 ± 3.3 bacteria:amoeba ratio) compared with E. aerogenes (3.96 ± 0.7 bacteria:amoeba ratio) and E. coli (5.85 ± 1.1 bacteria:amoeba ratio). A. hydrophila, E. coli, and E. aerogenes remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (14.13 ± 0.89 A. hydrophila:amoeba ratio, 10.13 ± 1.17 E. aerogenes:amoeba ratio, and 11.95 ± 0.7 E. coli:amoeba ratio). Conclusions A. hydrophila and E. aerogenes also joined the ranks of other bacteria that could benefit from A. castellanii

  17. Secretory expression of a heterologous nattokinase in Lactococcus lactis.

    PubMed

    Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

    2007-05-01

    Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

  18. Encapsulated Lactococcus lactis with enhanced gastrointestinal survival for the development of folate enriched functional foods.

    PubMed

    Divya, Jayakumar Beena; Nampoothiri, Kesavan Madhavan

    2015-01-01

    Two lactic acid bacteria (LAB) isolated from cow's milk were identified as Lactococcus lactis strains and designated as L. lactis CM22 and L. lactis CM28. They were immobilised by co-encapsulation using alginate and mannitol and by hybrid entrapment with skim milk, glycerol, CaCO3 and alginate. The encapsulated cells survived better in simulated gastrointestinal conditions compared to the free cells. The percentage survival of probiotics encapsulated by hybrid entrapment method was 62.74% for L. lactis CM22 and 68% for L. lactis CM28. Studies to check their efficacy in fermentative fortification of skim milk and ice cream revealed an enhancement in folate level.

  19. Development of a recombinant ureolytic Lactococcus lactis for urea removal.

    PubMed

    Zhang, Suai; Li, Dongxia; Tian, Kai; Bai, Yu; Zhang, Hongkai; Song, Cunjiang; Qiao, Mingqiang; Kong, Deling; Yu, Yaoting

    2009-01-01

    Kidney failure is a common disease with high frequency. Food-grade recombinant bacteria that can effectively remove urea has great potential for treatment of renal failure. A nonpathogenic strain, L. lactis MG1363, was transformed with plasmid pMG36eure, which carries urease gene. The expression of transgene urease in genetically modified L. lactis MG1363 and the urease activity in removal of urea were investigated. It was found that the removal of urea by recombinant L. lactis MG1363 was pH- and nickel-dependent. At pH 6.5 and in the presence of 250 microM of NiSO4, 50 approximately 60% of urea could be removed in 24 hours. The urea removal activity was also evaluated in imitative gastroenteric environment. After being exposed to acidic solution (pH2.5-4.0) for 2 hours, the cells were then grown in a medium containing 0.1 cfu/ml bile acid salt, 30 mg/dl urea, and 250 microM NiSO4 at pH 6.8. The concentration of urea decreased over time, and the removal was about 30% at 10 hours and 65% at 24 hours, respectively. The safety tests were performed by feeding normal rats with either L. lactis MG1363 or recombinant L. lactis MG1363. The two materials did not cause any changes in blood cells and blood biochemical indexes. There were no differences in terms of body weight and water/food consumption between the two materials. These results indicate the safety, feasibility, and capacity of urease gene modified Lactococcus Lactis in removal of urea under the gastroenteric circumstances. Further investigation may generate a food-grade strain for treatment of chronic renal failure.

  20. Characterization of PaxA and Its Operon: a Cohemolytic RTX Toxin Determinant from Pathogenic Pasteurella aerogenes

    PubMed Central

    Kuhnert, Peter; Heyberger-Meyer, Bénédicte; Nicolet, Jacques; Frey, Joachim

    2000-01-01

    Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times. PMID:10603361

  1. Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis

    PubMed Central

    Bemer, Pascale; Bodmer, Thomas; Munzinger, Juerg; Perrin, Monique; Vincent, Véronique; Drugeon, Henri

    2004-01-01

    The reliability of the MB/BACT system for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide, rifampin, isoniazid, streptomycin, and ethambutol was compared to the BACTEC 460TB system. The proportion method was used to resolve discrepant results by an independent arbiter. Two interpretative methods were used, with an undiluted control (direct control) and a diluted control (10−1 control). As no significant difference was observed between the two controls, the method with the direct control was adopted as the most accurate one. One hundred sixty-six strains were tested, with an overall agreement of 98.3%. After resolution of the 18 discrepant results by the proportion method, the sensitivity and specificity of the MB/BACT system were 100% for rifampin, isoniazid, and pyrazinamide. For ethambutol, sensitivity was 92.3% at the critical concentration and 33% at the high concentration, and specificity was 100% at both concentrations. For streptomycin, sensitivity was 100% at the critical concentration and 80% at the high concentration, and specificity was 98.6% at the critical concentration and 100% at the high concentration. The rifampin, isoniazid, streptomycin, and ethambutol susceptibility test results were obtained in 6.6 days with the MB/BACT versus 5 days with the BACTEC 460TB. The pyrazinamide susceptibility test results were obtained in 7.8 days with the MB/BACT, versus 6.7 days with the BACTEC 460TB. These data demonstrate that the fully automated MB/BACT system is a very reliable method for rapid susceptibility testing of M. tuberculosis against rifampin, isoniazid, and pyrazinamide. Sensitivity results have to be improved for ethambutol and streptomycin, especially at the high concentration. PMID:15004049

  2. Multicenter evaluation of the MB/BACT system for susceptibility testing of Mycobacterium tuberculosis.

    PubMed

    Bemer, Pascale; Bodmer, Thomas; Munzinger, Juerg; Perrin, Monique; Vincent, Véronique; Drugeon, Henri

    2004-03-01

    The reliability of the MB/BACT system for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide, rifampin, isoniazid, streptomycin, and ethambutol was compared to the BACTEC 460TB system. The proportion method was used to resolve discrepant results by an independent arbiter. Two interpretative methods were used, with an undiluted control (direct control) and a diluted control (10(-1) control). As no significant difference was observed between the two controls, the method with the direct control was adopted as the most accurate one. One hundred sixty-six strains were tested, with an overall agreement of 98.3%. After resolution of the 18 discrepant results by the proportion method, the sensitivity and specificity of the MB/BACT system were 100% for rifampin, isoniazid, and pyrazinamide. For ethambutol, sensitivity was 92.3% at the critical concentration and 33% at the high concentration, and specificity was 100% at both concentrations. For streptomycin, sensitivity was 100% at the critical concentration and 80% at the high concentration, and specificity was 98.6% at the critical concentration and 100% at the high concentration. The rifampin, isoniazid, streptomycin, and ethambutol susceptibility test results were obtained in 6.6 days with the MB/BACT versus 5 days with the BACTEC 460TB. The pyrazinamide susceptibility test results were obtained in 7.8 days with the MB/BACT, versus 6.7 days with the BACTEC 460TB. These data demonstrate that the fully automated MB/BACT system is a very reliable method for rapid susceptibility testing of M. tuberculosis against rifampin, isoniazid, and pyrazinamide. Sensitivity results have to be improved for ethambutol and streptomycin, especially at the high concentration.

  3. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143

  4. Multicenter evaluation of the MB/BACT system for susceptibility testing of Mycobacterium tuberculosis.

    PubMed

    Bemer, Pascale; Bodmer, Thomas; Munzinger, Juerg; Perrin, Monique; Vincent, Véronique; Drugeon, Henri

    2004-03-01

    The reliability of the MB/BACT system for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide, rifampin, isoniazid, streptomycin, and ethambutol was compared to the BACTEC 460TB system. The proportion method was used to resolve discrepant results by an independent arbiter. Two interpretative methods were used, with an undiluted control (direct control) and a diluted control (10(-1) control). As no significant difference was observed between the two controls, the method with the direct control was adopted as the most accurate one. One hundred sixty-six strains were tested, with an overall agreement of 98.3%. After resolution of the 18 discrepant results by the proportion method, the sensitivity and specificity of the MB/BACT system were 100% for rifampin, isoniazid, and pyrazinamide. For ethambutol, sensitivity was 92.3% at the critical concentration and 33% at the high concentration, and specificity was 100% at both concentrations. For streptomycin, sensitivity was 100% at the critical concentration and 80% at the high concentration, and specificity was 98.6% at the critical concentration and 100% at the high concentration. The rifampin, isoniazid, streptomycin, and ethambutol susceptibility test results were obtained in 6.6 days with the MB/BACT versus 5 days with the BACTEC 460TB. The pyrazinamide susceptibility test results were obtained in 7.8 days with the MB/BACT, versus 6.7 days with the BACTEC 460TB. These data demonstrate that the fully automated MB/BACT system is a very reliable method for rapid susceptibility testing of M. tuberculosis against rifampin, isoniazid, and pyrazinamide. Sensitivity results have to be improved for ethambutol and streptomycin, especially at the high concentration. PMID:15004049

  5. Production of gluconic acid and 2-ketogluconic acid by Klebsiella aerogenes NCTA 418.

    PubMed

    Neijssel, O M; Tempest, D W

    1975-10-27

    2-Ketogluconic acid and, to a lesser extent, gluconic acid were found to be major products of glucose catabolism by phosphate-limited cultures of Klebsiella aerogenes NCTC 418, and together accounted for up to 46% of the glucose carbon that was metabolized. Although the concentrations of both acids increased substantially at low growth rates, their specific rates of synthesis decreased markedly, ad did the proportion of glucose converted into these products. Determination of the affinity constant, for glucose, of phosphate-limited organisms showed it ot be not significantly different from that of glucose-limited organisms (KS less than or equal to 50 muM), indicative of the phosphotransferase uptake system. And since these organisms possessed an active glucose 6-phosphate dehydrogenase, and had no detectable glucose dehydrogenase activity, it was concluded that gluconic acid and 2-keto-gluconic acid arose from their corresponding phosphorylated metabolites, and not directly from glucose.

  6. Biodegradation of dichlorodiphenyltrichloroethane: intermediates in dichlorodiphenylacetic acid metabolism by aerobacter aerogenes

    USGS Publications Warehouse

    Wedemeyer, Gary

    1967-01-01

    The final product of dichlorodiphenyltrichloroethane (DDT) degradation by vertebrates is commonly considered to be dichlorodiphenylacetic acid, DDA. Recently, certain organisms have been found to degrade further DDA to dichlorobenzophenone (DBP), but the possibility that such degradation was due to microbial action could not be excluded. Significantly, dichlorobenzhydrol (DBH), dichlorophenylmethane (DPM), and dichlorodiphenylethylene (DDE) have been tentatively identified in rats fed DDA. Since DDA as well as DDT is degraded by the ubiquitous microorganism Aerobacter aerogenes, it seemed reasonable that the intestinal microflora might be involved in DBP formation, DPM and DBH being intermediates in its pathway from DDA. Since DDA is a (3,y-unsaturated acid, ketone formation via an alkene and an alcohol would be expected.

  7. Polygalacturonase production by calcium alginate immobilized Enterobacter aerogenes NBO2 cells.

    PubMed

    Darah, I; Nisha, M; Lim, Sheh-Hong

    2015-03-01

    Bacterial cells of Enterobacter aerogenes NBO2 were entrapped in calcium alginate beads in order to enhance polygalacturonase production compared to free cells. The optimized condition of 5 % (w/v) sodium alginate concentration, agitation speed of 250 rpm, and 15 beads of calcium alginate with inoculum size of 4 % (v/v; 5.4 × 10(7) cells/ml) produced 23.48 U/mL of polygalacturonase compared to free cells of 18.54 U/ml. There was about 26.6 % increment in polygalaturonase production. However, in this study, there was 296.6 % of increment in polygalacturonase production after improvement parameters compared to before improvement parameters of calcium alginate bead immobilization cells (5.92 U/ml). This research has indicated that optimized physical parameters of calcium alginate bead immobilization cells have significantly enhanced the production of polygalacturonase.

  8. Blood infection with Enterobacter aerogenes--an unusual cause of portal vein gas.

    PubMed

    Fayyaz, Afshan

    2011-01-01

    Portal vein gas was once thought of as an invariably fatal condition. Now, with the availability of better equipment and expertise, the condition is more frequently diagnosed. A case of fever with rigors is presented and on ultrasound and CT examination was found to have portal venous gas which resolved with adequate antibiotic treatment. Blood culture revealed growth of gram negative bacillus; Enterobacter aerogenes. Patient was investigated further for portal vein gas, and although no other cause for the development of portal vein gas was found, she was treated with antibiotics and showed an immediate response. The aim of this case report is to highlight the benign causes of portal vein gas as well as to discuss the causes which warrant immediate surgery. Portal vein gas may herald a more ominous condition, which if intercepted in its course may result in complete cure.

  9. Irreproducible and uninterpretable Polymyxin B MICs for Enterobacter cloacae and Enterobacter aerogenes.

    PubMed

    Landman, David; Salamera, Julius; Quale, John

    2013-12-01

    Carbapenem-resistant Enterobacter species are emerging nosocomial pathogens. As with most multidrug-resistant Gram-negative pathogens, the polymyxins are often the only therapeutic option. In this study involving clinical isolates of E. cloacae and E. aerogenes, susceptibility testing methods with polymyxin B were analyzed. All isolates underwent testing by the broth microdilution (in duplicate) and agar dilution (in duplicate) methods, and select isolates were examined by the Etest method. Selected isolates were also examined for heteroresistance by population analysis profiling. Using a susceptibility breakpoint of ≤2 μg/ml, categorical agreement by all four dilution tests (two broth microdilution and two agar dilution) was achieved in only 76/114 (67%) of E. cloacae isolates (65 susceptible, 11 resistant). Thirty-eight (33%) had either conflicting or uninterpretable results (multiple skip wells, i.e., wells that exhibit no growth although growth does occur at higher concentrations). Of the 11 consistently resistant isolates, five had susceptible MICs as determined by Etest. Heteroresistant subpopulations were detected in eight of eight isolates tested, with greater percentages in isolates with uninterpretable MICs. For E. aerogenes, categorical agreement between the four dilution tests was obtained in 48/56 (86%), with conflicting and/or uninterpretable results in 8/56 (14%). For polymyxin susceptibility testing of Enterobacter species, close attention must be paid to the presence of multiple skip wells, leading to uninterpretable results. Susceptibility also should not be assumed based on the results of a single test. Until the clinical relevance of skip wells is defined, interpretation of polymyxin susceptibility tests for Enterobacter species should be undertaken with extreme caution.

  10. Biosensing and bioremediation of Cr(VI) by cell free extract of Enterobacter aerogenes T2.

    PubMed

    Panda, Jigisha; Sarkar, Priyabrata

    2014-01-01

    Hexavalent chromium or Cr(VI) enters the environment through several anthropogenic activities and it is highly toxic and carcinogenic. Hence it is required to be detected and remediated from the environment. In this study, low-cost and environment-friendly methods of biosensing and bioremediation of Cr(VI) have been proposed. Crude cell free extract (CFE) of previously isolated Enterobacter aerogenes T2 (GU265554; NII 1111) was prepared and exploited to develop a stable biosensor for direct estimation of Cr(VI) in waste water, by using three electrodes via cyclic voltammetry. For bioremediation studies, a homogeneous solution of commercially available sodium alginate and CFE was added dropwise in a continuously stirred calcium chloride solution. Biologically modified calcium alginate beads were produced and these were further utilized for bioremediation studies. The proposed sensor showed linear response in the range of 10-40 μg L(-1) Cr(VI) and the limit of detection was found to be 6.6 μg L(-1) Cr(VI). No interference was observed in presence of metal ions, e.g., lead, cadmium, arsenic, tin etc., except for insignificant interference with molybdenum and manganese. In bioremediation studies, modified calcium alginate beads showed encouraging removal rate 900 mg Cr(VI)/m(3) water per day with a removal efficiency of 90%, much above than reported in literature. The proposed sensing system could be a viable alternative to costly measurement procedures. Calcium alginate beads, modified with CFE of E. aerogenes, could be used in bioremediation of Cr(VI) since it could work in real conditions with extraordinarily high capacity.

  11. Cloning and expression of a novel lactococcal aggregation factor from Lactococcus lactis subsp. lactis BGKP1

    PubMed Central

    2011-01-01

    Background Aggregation may play a main role in the adhesion of bacteria to the gastrointestinal epithelium and their colonization ability, as well as in probiotic effects through co-aggregation with intestinal pathogens and their subsequent removal. The aggregation phenomenon in lactococci is directly associated with the sex factor and lactose plasmid co-integration event or duplication of the cell wall spanning (CWS) domain of PrtP proteinase. Results Lactococcus lactis subsp. lactis BGKP1 was isolated from artisanal semi-hard homemade cheese and selected due to its strong auto-aggregation phenotype. Subsequently, non-aggregating derivative (Agg-) of BGKP1, designated as BGKP1-20, was isolated, too. Comparative analysis of cell surface proteins of BGKP1 and derivative BGKP1-20 revealed a protein of approximately 200 kDa only in the parental strain BGKP1. The gene involved in aggregation (aggL) was mapped on plasmid pKP1 (16.2 kb), cloned and expressed in homologous and heterologous lactococci and enterococci. This novel lactococcal aggregation protein was shown to be sufficient for cell aggregation in all tested hosts. In addition to the aggL gene, six more ORFs involved in replication (repB and repX), restriction and modification (hsdS), transposition (tnp) and possible interaction with mucin (mbpL) were also located on plasmid pKP1. Conclusion AggL is a new protein belonging to the collagen-binding superfamily of proteins and is sufficient for cell aggregation in lactococci. PMID:22182285

  12. Recombinant Lactococcus lactis fails to secrete bovine chymosine

    PubMed Central

    Luerce, Tessália Diniz; Azevedo, Marcela Santiago Pacheco; LeBlanc, Jean Guy; Azevedo, Vasco; Miyoshi, Anderson; Pontes, Daniela Santos

    2014-01-01

    Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein. PMID:25482140

  13. Lactose-mediated carbon catabolite repression of putrescine production in dairy Lactococcus lactis is strain dependent.

    PubMed

    del Rio, Beatriz; Ladero, Victor; Redruello, Begoña; Linares, Daniel M; Fernández, Maria; Martín, Maria Cruz; Alvarez, Miguel A

    2015-06-01

    Lactococcus lactis is the lactic acid bacterial (LAB) species most widely used as a primary starter in the dairy industry. However, several strains of L. lactis produce the biogenic amine putrescine via the agmatine deiminase (AGDI) pathway. We previously reported the putrescine biosynthesis pathway in L. lactis subsp. cremoris GE2-14 to be regulated by carbon catabolic repression (CCR) via glucose but not lactose (Linares et al., 2013). The present study shows that both these sugars repress putrescine biosynthesis in L. lactis subsp. lactis T3/33, a strain isolated from a Spanish artisanal cheese. Furthermore, we demonstrated that both glucose and lactose repressed the transcriptional activity of the aguBDAC catabolic genes of the AGDI route. Finally, a screening performed in putrescine-producing dairy L. lactis strains determined that putrescine biosynthesis was repressed by lactose in all the L. lactis subsp. lactis strains tested, but in only one L. lactis subsp. cremoris strain. Given the obvious importance of the lactose-repression in cheese putrescine accumulation, it is advisable to consider the diversity of L. lactis in this sense and characterize consequently the starter cultures to select the safest strains.

  14. Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli.

    PubMed Central

    Bojovic, B; Djordjevic, G; Banina, A; Topisirovic, L

    1994-01-01

    Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression. Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis. These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the secondary structure of the cat mRNA. New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning. PMID:7961430

  15. Statistical optimization of cell disruption techniques for releasing intracellular X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis spp. lactis.

    PubMed

    Üstün-Aytekin, Özlem; Arısoy, Sevda; Aytekin, Ali Özhan; Yıldız, Ece

    2016-03-01

    X-prolyl dipeptidyl aminopeptidase (PepX) is an intracellular enzyme from the Gram-positive bacterium Lactococcus lactis spp. lactis NRRL B-1821, and it has commercial importance. The objective of this study was to compare the effects of several cell disruption methods on the activity of PepX. Statistical optimization methods were performed for two cavitation methods, hydrodynamic (high-pressure homogenization) and acoustic (sonication), to determine the more appropriate disruption method. Two level factorial design (2FI), with the parameters of number of cycles and pressure, and Box-Behnken design (BBD), with the parameters of cycle, sonication time, and power, were used for the optimization of the high-pressure homogenization and sonication methods, respectively. In addition, disruption methods, consisting of lysozyme, bead milling, heat treatment, freeze-thawing, liquid nitrogen, ethylenediaminetetraacetic acid (EDTA), Triton-X, sodium dodecyl sulfate (SDS), chloroform, and antibiotics, were performed and compared with the high-pressure homogenization and sonication methods. The optimized values of high-pressure homogenization were one cycle at 130 MPa providing activity of 114.47 mU ml(-1), while sonication afforded an activity of 145.09 mU ml(-1) at 28 min with 91% power and three cycles. In conclusion, sonication was the more effective disruption method, and its optimal operation parameters were manifested for the release of intracellular enzyme from a L. lactis spp. lactis strain, which is a Gram-positive bacterium. PMID:26584994

  16. Statistical optimization of cell disruption techniques for releasing intracellular X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis spp. lactis.

    PubMed

    Üstün-Aytekin, Özlem; Arısoy, Sevda; Aytekin, Ali Özhan; Yıldız, Ece

    2016-03-01

    X-prolyl dipeptidyl aminopeptidase (PepX) is an intracellular enzyme from the Gram-positive bacterium Lactococcus lactis spp. lactis NRRL B-1821, and it has commercial importance. The objective of this study was to compare the effects of several cell disruption methods on the activity of PepX. Statistical optimization methods were performed for two cavitation methods, hydrodynamic (high-pressure homogenization) and acoustic (sonication), to determine the more appropriate disruption method. Two level factorial design (2FI), with the parameters of number of cycles and pressure, and Box-Behnken design (BBD), with the parameters of cycle, sonication time, and power, were used for the optimization of the high-pressure homogenization and sonication methods, respectively. In addition, disruption methods, consisting of lysozyme, bead milling, heat treatment, freeze-thawing, liquid nitrogen, ethylenediaminetetraacetic acid (EDTA), Triton-X, sodium dodecyl sulfate (SDS), chloroform, and antibiotics, were performed and compared with the high-pressure homogenization and sonication methods. The optimized values of high-pressure homogenization were one cycle at 130 MPa providing activity of 114.47 mU ml(-1), while sonication afforded an activity of 145.09 mU ml(-1) at 28 min with 91% power and three cycles. In conclusion, sonication was the more effective disruption method, and its optimal operation parameters were manifested for the release of intracellular enzyme from a L. lactis spp. lactis strain, which is a Gram-positive bacterium.

  17. Exploring "aerogen-hydride" interactions between ZOF2 (Z = Kr, Xe) and metal hydrides: An ab initio study

    NASA Astrophysics Data System (ADS)

    Esrafili, Mehdi D.; Mohammadian-Sabet, Fariba

    2016-06-01

    In this work, a new σ-hole interaction formed between ZOF2 (Z = Kr and Xe) as the Lewis acid and a series of metal-hydrides HMX (M = Be, Mg, Zn and X = H, F, CN, CH3) is reported. The nature of this interaction, called "aerogen-hydride" interaction, is unveiled by molecular electrostatic potential, non-covalent interaction, quantum theory of atoms in molecules and natural bond orbital analyses. Our results indicate that the aerogen-hydride interactions are quite strong and can be comparable in strength to other σ-hole bonds. An important charge-transfer interaction is also associated with the formation of OF2Z⋯HMX complexes.

  18. KPC-2 carbapenemase and DHA-1 AmpC determinants carried on the same plasmid in Enterobacter aerogenes.

    PubMed

    Kuai, Shougang; Shao, Haifeng; Huang, Lihua; Pei, Hao; Lu, Zhonghua; Wang, Weiping; Liu, Jun

    2014-03-01

    This study was conducted to analyse the presence of a plasmid-mediated carbapenem resistance mechanism in a clinical Enterobacter aerogenes isolate from a patient from Jiangsu province, People's Republic of China. PCR and sequencing confirmed that the isolate harboured Klebsiella pneumoniae carbapenemase (KPC)-2, DHA-1 and TEM-1 β-lactamase genes. Both the KPC-2 and DHA-1 genes were transferred to Escherichia coli C600 by transconjugation, and Southern blotting confirmed that these two genes were located on the same plasmid, which was of approximately 56 kb in size. The Enterobacter aerogenes isolate was resistant to carbapenems and other tested antimicrobial agents. The Escherichia coli transconjugant showed reduced susceptibility but not resistance to carbapenems and other β-lactams, indicating the presence of another, possibly permeability-related, resistance mechanism in the clinical isolate.

  19. Co-fermentation of carbon sources by Enterobacter aerogenes ATCC 29007 to enhance the production of bioethanol.

    PubMed

    Thapa, Laxmi Prasad; Lee, Sang Jun; Yang, Xiao Guang; Yoo, Hah Young; Kim, Sung Bong; Park, Chulhwan; Kim, Seung Wook

    2014-06-01

    We investigated the enhancement of bioethanol production in Enterobacter aerogenes ATCC 29007 by co-fermentation of carbon sources such as glycerol, glucose, galactose, sucrose, fructose, xylose, starch, mannitol and citric acid. Biofuel production increases with increasing growth rate of microorganisms; that is why we investigated the optimal growth rate of E. aerogenes ATCC 29007, using mixtures of different carbon sources with glycerol. E. aerogenes ATCC 29007 was incubated in media containing each carbon source and glycerol; growth rate and bioethanol production improved in all cases compared to those in medium containing glycerol alone. The growth rate and bioethanol production were highest with mannitol. Fermentation was carried out at 37 °C for 18 h, pH 7, using 50 mL defined production medium in 100 mL serum bottles at 200 rpm. Bioethanol production under optimized conditions in medium containing 16 g/L mannitol and 20 g/L glycerol increased sixfold (32.10 g/L) than that containing glycerol alone (5.23 g/L) as the carbon source in anaerobic conditions. Similarly, bioethanol production using free cells in continuous co-fermentation also improved (27.28 g/L) when 90.37 % of 16 g/L mannitol and 67.15 % of 20 g/L glycerol were used. Although naturally existing or engineered microorganisms can ferment mixed sugars sequentially, the preferential utilization of glucose to non-glucose sugars often results in lower overall yield and productivity of ethanol. Here, we present new findings in E. aerogenes ATCC 29007 that can be used to improve bioethanol production by simultaneous co-fermentation of glycerol and mannitol.

  20. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis.

    PubMed

    Kobierecka, Patrycja A; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M; Jagusztyn-Krynicka, Elżbieta K; Wyszyńska, Agnieszka K

    2016-01-01

    Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein - CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to

  1. Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis

    PubMed Central

    Kobierecka, Patrycja A.; Olech, Barbara; Książek, Monika; Derlatka, Katarzyna; Adamska, Iwona; Majewski, Paweł M.; Jagusztyn-Krynicka, Elżbieta K.; Wyszyńska, Agnieszka K.

    2016-01-01

    Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type C. jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analyzed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ Lactic Acid Bacteria (LAB) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to

  2. Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

    SciTech Connect

    Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

    1986-02-01

    Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.

  3. Generation of dipeptidyl peptidase-IV-inhibiting peptides from β-lactoglobulin secreted by Lactococcus lactis.

    PubMed

    Shigemori, Suguru; Oshiro, Kazushi; Wang, Pengfei; Yamamoto, Yoshinari; Wang, Yeqin; Sato, Takashi; Uyeno, Yutaka; Shimosato, Takeshi

    2014-01-01

    Previous studies showed that hydrolysates of β-lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus. PMID:25157356

  4. Characterization of a Wild, Novel Nisin A-Producing Lactococcus Strain with an L. lactis subsp. cremoris Genotype and an L. lactis subsp. lactis Phenotype, Isolated from Greek Raw Milk

    PubMed Central

    Parapouli, Maria; Delbès-Paus, Céline; Kakouri, Athanasia; Koukkou, Anna-Irini; Montel, Marie-Christine

    2013-01-01

    Several molecular taxonomic studies have revealed that many natural (wild) Lactococcus lactis strains of dairy origin which are phenotypically representative of the L. lactis subspecies lactis cluster genotypically within subspecies cremoris and vice versa. Recently, we isolated two wild nisin-producing (Nis+) L. lactis strains, M78 and M104, of the lactis phenotype from Greek raw milk (J. Samelis, A. Lianou, A. Kakouri, C. Delbès, I. Rogelj, B. B. Matijašic, and M. C. Montel, J. Food Prot. 72:783–790, 2009); strain M78 possess a novel nisin A sequence (GenBank accession number HM219853). In this study, the actual subspecies identity of M78 and M104 isolates was elucidated, using 16S rRNA and acmA (encoding lactococcal N-acetylmuramidase) gene and histidine biosynthesis operon polymorphisms and 16S rRNA and ldh (encoding lactate dehydrogenase) gene phylogenies. Except the acmA gene analysis, molecular tools revealed that isolates M78 and M104 clustered with strains of the cremoris genotype, including the LMG 6897T strain, while they were distant from strains of the lactis genotype, including the LMG 6890T strain. The two wild isolates had identical repetitive sequence-based PCR (rep-PCR), randomly amplified polymorphic DNA (RAPD), plasmid, and whole-cell protein profiles and shared high 16S rRNA (99.9%) and ldh (100%) gene sequence homologies. In contrast, they exhibited identical sugar fermentation and enzymatic patterns which were similar to those of the subspecies lactis LMG 6890T strain. To our knowledge, this is the first complete identification report on a wild L. lactis subsp. cremoris genotype of the lactis phenotype which is capable of nisin A production and, thus, has strong potential for use as a novel dairy starter and/or protective culture. PMID:23542625

  5. Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes.

    PubMed

    Fulks, R M; Stadtman, E R

    1985-12-13

    When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but

  6. Lactococcus lactis-based vaccines from laboratory bench to human use: an overview.

    PubMed

    Bahey-El-Din, Mohammed

    2012-01-17

    Developing effective vaccines is an important weapon in the battle against potential pathogens and their evolving antibiotic resistance trends. Several vaccine delivery vectors have been investigated among which the generally regarded as safe (GRAS) Lactococcus lactis has a distinguished position. In this review, different factors affecting the efficacy of L. lactis-based vaccines are discussed. In addition, the issues of biological containment and pharmaceutical quality assurance of L. lactis vaccines are highlighted. These issues are critical for the success of medical translation of L. lactis-based vaccines from research laboratories to clinical use by ensuring consistent manufacturing of safe and efficacious vaccines.

  7. Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

    PubMed Central

    Millen, Anne M.; Horvath, Philippe; Boyaval, Patrick; Romero, Dennis A.

    2012-01-01

    Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes. PMID:23240053

  8. Proteomic Signature of Lactococcus lactis NCDO763 Cultivated in Milk†

    PubMed Central

    Gitton, Christophe; Meyrand, Mickael; Wang, Juhui; Caron, Christophe; Trubuil, Alain; Guillot, Alain; Mistou, Michel-Yves

    2005-01-01

    We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment. PMID:16269754

  9. Les biofilms bactériens : leur importance en santé animale et en santé publique

    PubMed Central

    Tremblay, Yannick D.N.; Hathroubi, Skander; Jacques, Mario

    2014-01-01

    Résumé Les biofilms bactériens sont des amas structurés de cellules bactériennes enrobés d’une matrice polymérique et attachés à une surface. Le biofilm protège les bactéries et leur permet de survivre dans des conditions environnementales hostiles. Les bactéries du biofilm peuvent résister à la réponse immunitaire de l’hôte et sont beaucoup plus résistantes aux antibiotiques et aux désinfectants que les cellules bactériennes planctoniques. La capacité de former un biofilm est maintenant reconnue comme une caractéristique propre à plusieurs microorganismes. La présence de biofilms lors d’infections demande donc de nouvelles méthodes de prévention, de diagnostic et de traitement. De même, la présence de biofilms sur des surfaces retrouvées à la ferme, à l’abattoir ou à l’usine de transformation affectera l’efficacité du protocole de désinfection. De façon surprenante, la formation de biofilms chez les bactéries pathogènes des animaux et les bactéries zoonotiques est un sujet relativement peu étudié. Ce bref compte rendu a pour objectif de sensibiliser les intervenants en santé animale à l’importance des biofilms. PMID:24688172

  10. Growth of Enterobacter aerogenes in a chemostat with double nutrient limitations.

    PubMed Central

    Cooney, C L; Wang, D I; Mateles, R I

    1976-01-01

    The behavior of Enterobacter aerogenes during growth in chemostats limited by single and double nutrient restrictions was examined. On the assumption that different essential nutrients act to limit growth in different ways, we selected pairs of nutrients likely to affect different aspects of metabolism. Results show that macromolecular cell composition can be controlled by using more than one nutrient restriction. The polysaccharide content of the cells is readily manipulated by the ratio of carbon to nitrogen in the inlet nutrients. Also, at low dilution rates, ratios of protein to ribonucleic acid are dependent on the ratio of phosphate to nitrogen in the input nutrients. An examination of both acetic acid and metabolite production (as measured by ultraviolet absorbance of culture filtrates) showed that accumulation of these products was dependent on both dilution rate and type of nutrient limitation(s). These results were examined in terms of the problems of translation of batch to continuous culture processes and the use of selected nutrient limitations to control noncellular product formation. PMID:942212

  11. Aerogenic vaccination with a Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice.

    PubMed

    Ulrich, Ricky L; Amemiya, Kei; Waag, David M; Roy, Chad J; DeShazer, David

    2005-03-14

    Burkholderia mallei is an obligate mammalian pathogen that causes the zoonotic disease glanders. Two live attenuated B. mallei strains, a capsule mutant and a branched-chain amino acid auxotroph, were evaluated for use as vaccines against aerosol-initiated glanders in mice. Animals were aerogenically vaccinated and serum samples were obtained before aerosol challenge with a high-dose (>300 times the LD50) of B. mallei ATCC 23344. Mice vaccinated with the capsule mutant developed a Th2-like Ig subclass antibody response and none survived beyond 5 days. In comparison, the auxotrophic mutant elicited a Th1-like Ig subclass antibody response and 25% of the animals survived for 1 month postchallenge. After a low-dose (5 times the LD50) aerosol challenge, the survival rates of auxotroph-vaccinated and unvaccinated animals were 50 and 0%, respectively. Thus, live attenuated strains that promote a Th1-like Ig response may serve as promising vaccine candidates against aerosol infection with B. mallei.

  12. Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme.

    PubMed Central

    Lee, M H; Mulrooney, S B; Hausinger, R P

    1990-01-01

    Urease was purified from recombinant Klebsiella aerogenes which was grown in the absence of nickel. The protein was inactive and contained no transition metals, yet it possessed the same heteropolymeric structure as native enzyme, demonstrating that Ni is not required for intersubunit association. Ni did, however, substantially increase the stability of the intact metalloprotein (Tm = 79 degrees C) compared with apoenzyme (Tm = 62 degrees C), as revealed by differential scanning calorimetric analysis. An increased number of histidine residues were accessible to diethyl pyrocarbonate in apourease compared with holoenzyme, consistent with possible Ni ligation by histidinyl residues. Addition of Ni to purified apourease did not yield active enzyme; however, urease apoenzyme was very slowly activated in vivo by addition of Ni ions to Ni-free cell cultures, even after treatment of the cells with spectinomycin to inhibit protein synthesis. In contrast, sonicated cells and cells treated with dinitrophenol or dicyclohexylcarbodiimide were incapable of activating apourease. These results indicate that apourease activation is an energy-dependent process that is destroyed by cell disruption. PMID:2142939

  13. Growth and heavy metal removal by Klebsiella aerogenes at different pH and temperature

    SciTech Connect

    Al-Shahwani, M.F.; Jazrawi, S.F.; Al-Rawi, E.H.; Ayar, N.S.

    1984-01-01

    A strain of Klebsiella aerogenes isolated from Rustamiyah Station for treatment of wastewater was examined for its ability to grow in a media supplemented with maximum tolerance concentrations of Pb/sup + +/, Zn/sup + +/, Ni/sup + +/, and Cd/sup + +/, separately, at different temperatures and initial pH. The results indicated that at 28/sup 0/C during the first 24 hr, Pb/sup + +/ and Ni/sup + +/ had no effect on the growth of the bacteria, while the presence of Zn/sup + +/ and Cd/sup + +/ decreased the cell count. The growth reached a maximum level after the second day and started to decrease gradually. The bacterial count at 37/sup 0/C was less than that at 28/sup 0/C. No bacterial multiplication occurred at 44/sup 0/C. There was little difference between heavy metal removal at 28 and 37/sup 0/C. At 44/sup 0/C, little removal took place. In general, slightly acidic or neutral medium was better for both bacterial growth and metal removal.

  14. Detoxification of mercury, cadmium, and lead in Klebsiella aerogenes NCTC 418 growing in continuous culture

    SciTech Connect

    Aiking, H.; Govers, H.; van 'T Riet, J.

    1985-11-01

    Klebsiella aerogenes NCTC 418 growing in the presence of cadmium under glucose-, sulfate-, or phosphate-limited conditions in continuous culture exhibited sulfide formation and P/sub i/ accumulation as the only demonstrable detoxification mechanisms. In the presence of mercury under similar conditions only HgS formation could be confirmed, by an increased sensitivity to mercury under sulfate-limited conditions, among others. The fact that the cells were most sensitive to cadmium under conditions of phosphate limitation and most sensitive to mercury under conditions of sulfate limitation led to the hypothesis that these inorganic detoxification mechanisms generally depended on a kind of facilitated precipitation. The process was coined thus because heavy metals were probably accumulated and precipitated near the cell perimeter due to the relatively high local concentrations of sulfide and phosphate there. Depending on the growth-limiting nutrient, mercury proved to be 25-fold (phosphate limitation), 75-fold (glycerol limitation), or 150-fold (sulfate limitation) more toxic than cadmium to this organism. In the presence of lead, PbS formation was suggested. since no other detoxification mechanisms were detected, for example, rendering heavy metal ions innocuous as metallo-organic compounds, it was concluded that formation of heavy metal precipitates is crucially important to this organism. In addition, it was observed that several components of a defined mineral medium were able to reduce mercuric ions to elemental mercury. This abiotic mercury volatilization was studied in detail, and its general and environmental implications are discussed.

  15. Biodegradation of ichlorodiphenyltrichloroe-thane: Intermediates in dichlorodiphenylacetic acid metabolism by Aerobacter aerogenes

    USGS Publications Warehouse

    1967-01-01

    The final product of dichlorodiphenyltrichloroethane (DDT) degradation by vertebrates is commonly considered to be dichlorodiphenylacetic acid, DDA (J. E. Peterson and W. H. Robison, Toxicol. Appl. Pharmacol. 6:321, 1964). Recently, certain organisms (A. S. Perry, S. Miller, and A. J. Buckner. J. Agr. Food Chem. 11:457, 1963; J. D. Pinto, M. N. Comien, and M. S. Dunn. J. Biol. Chem. 240:2148, 1965) have been found to degrade further DDA to dichlorobenzophenone (DBP), but the possibility that such degradation was due to microbial action could not be excluded. Significantly, dichlorobenzhydrol (DBH), dichlorophenylmethane (DPM), and dichlorodiphenylethylene (DDE) have been tentatively identified in rats fed DDA (Pinto et al., J. Biol. Chem. 240:2148, 1965). Since DDA as well as DDT is degraded by the ubiquitous microorganism Aerobacter aerogenes (G. Wedemeyer, Appl. Microbiol. 15:569, 1967; J. L. Mendel, and M. S. Walton, Science 151:1527, 1966), it seemed reasonable that the intestinal microflora might be involved in DBP formation, DPM and DBH being intermediates in its pathway from DDA. Since DDA is a (3,y-unsaturated acid, ketone formation via an alkene and an alcohol would be expected (S. G. Waley, Mechanisms of Organic and Enzymatic Reactions, Oxford University Press, London, England 1962).

  16. Optimization of cultural conditions for conversion of glycerol to ethanol by Enterobacter aerogenes S012

    PubMed Central

    2013-01-01

    The aim of this research is to optimize the cultural conditions for the conversion of glycerol to ethanol by Enterobacter aerogenes S012. Taguchi method was used to screen the cultural conditions based on their signal to noise ratio (SN). Temperature (°C), agitation speed (rpm) and time (h) were found to have the highest influence on both glycerol utilization and ethanol production by the organism while pH had the lowest. Full factorial design, statistical analysis, and regression model equation were used to optimize the selected cultural parameters for maximum ethanol production. The result showed that fermentation at 38°C and 200 rpm for 48 h would be ideal for the bacteria to produce maximum amount of ethanol from glycerol. At these optimum conditions, ethanol production, yield and productivity were 25.4 g/l, 0.53 g/l/h, and 1.12 mol/mol-glycerol, repectively. Ethanol production increased to 26.5 g/l while yield and productivity decreased to 1.04 mol/mol-glycerol and 0.37 g/l/h, respectively, after 72 h. Analysis of the fermentation products was performed using HPLC, while anaerobic condition was created by purging the fermentation vessel with nitrogen gas. PMID:23388539

  17. Effect of crude glycerol-derived inhibitors on ethanol production by Enterobacter aerogenes.

    PubMed

    Lee, Sang Jun; Kim, Sung Bong; Kang, Seong Woo; Han, Sung Ok; Park, Chulhwan; Kim, Seung Wook

    2012-01-01

    In this study, ethanol production from pure and crude glycerol using Enterobacter aerogenes ATCC 29007 was evaluated under anaerobic culture conditions. Inhibitory effects of substrate concentrations, pH, and salt concentrations were investigated based on crude glycerol components. Ethanol production was performed with pure glycerol concentrations ranging from 5 to 30 g/L to evaluate the effects of substrate concentration and osmotic pressure. The consumed glycerol was 5-14.33 g/L, and the yield of ethanol was higher than 0.75 mol ethanol/mol glycerol after 24 h of cultivation. To evaluate the inhibitory effects of salts (NaCl and KCl), experiments were performed with 0-20 g/L of each salt. Inhibitory effects of salts were strongest at high salt concentrations. The inhibitory effect of pH was performed in the pH range 4-10, and cell growth and ethanol production were highest at pH 5-6. Also, ethanol production was slightly inhibited at low concentration of crude glycerol comparison with pure glycerol. However, significant inhibitory effects were not observed at 1.5 and 2% crude glycerol which showed higher ethanol production compared to pure glycerol.

  18. Biodegradation of acrylamide by Enterobacter aerogenes isolated from wastewater in Thailand.

    PubMed

    Buranasilp, Kanokhathai; Charoenpanich, Jittima

    2011-01-01

    A widespread use of acrylamide, probably a neurotoxicant and carcinogen, in various industrial processes has led to environmental contamination. Fortunately, some microorganisms are able to derive energy from acrylamide. In the present work, we reported the isolation and characterization of a novel acrylamide-degrading bacterium from domestic wastewater in Chonburi, Thailand. The strain grew well in the presence of acrylamide as 0.5% (W/V), at pH 6.0 to 9.0 and 25 degrees C. Identification based on biochemical characteristics and 16S rRNA gene sequence identified the strain as Enterobacter aerogenes. Degradation of acrylamide to acrylic acid started in the late logarithmic growth phase as a biomass-dependent pattern. Specificity of cell-free supernatant towards amides completely degraded butyramide and urea and 86% of lactamide. Moderate degradation took place in other amides with that by formamide > benzamide > acetamide > cyanoacetamide > propionamide. No degradation was detected in the reactions of N,N-methylene bisacrylamide, sodium azide, thioacetamide, and iodoacetamide. These results highlighted the potential of this bacterium in the cleanup of acrylamide/amide in the environment.

  19. Improved hydrogen production under microaerophilic conditions by overexpression of polyphosphate kinase in Enterobacter aerogenes.

    PubMed

    Lu, Yuan; Zhang, Chong; Lai, Qiheng; Zhao, Hongxin; Xing, Xin-Hui

    2011-02-01

    Effects of different microaerophilic conditions on cell growth, glucose consumption, hydrogen production and cellular metabolism of wild Enterobacter aerogenes strain and polyphosphate kinase (PPK) overexpressing strain were systematically studied in this paper, using NaH(2)PO(4) as the phosphate sources. Under different microaerophilic conditions, PPK-overexpressing strain showed better cell growth, glucose consumption and hydrogen production than the wild strain. In the presence of limited oxygen (2.1%) and by PPK overexpression, the hydrogen production per liter of culture, the hydrogen production per cell and the hydrogen yield per mol of glucose increased by 20.1%, 12.3% and 10.8%, respectively, compared with the wild strain under strict anaerobic conditions. Metabolic analysis showed that the increase of the total hydrogen yield was attributed to the improvement of NADH pathway. The result of more reductive cellular oxidation state balance also further demonstrated that, under proper initial microaerophilic conditions and by PPK overexpression, the cell could adjust the cellular redox states and make more energy flow into hydrogen production pathways.

  20. [Mechanisms of bioelectricity generation in Enterobacter aerogenes-based microbial fuel cells].

    PubMed

    Zhang, Jin-Tao; Zhou, Shun-Gui; Zhang, Li-Xia; Lu, Na; Deng, Li-Fang; Ni, Jin-Ren

    2009-04-15

    Microbial fuel cells (MFCs) using hydrogen-producing bacteria (HPB) could utilize a large number of substrates to generate power. However, the coulombic efficiency is limited by the fact that only suspended cells are used as biocatalyst in anodic medium. MFCs using Fe (III)-reducing bacteria have high energy recovery efficiency, but can only utilize some simple organic matters. In this study, Enterobacter aerogenes XM02, a hydrogen-producing strain with Fe(III)-reducing activity, was selected as biocatalyst for MFCs, which could produce electricity by digesting lots of carbohydrates even starch. Graphite felt, a material with high specific surface area and hydrogen catalysis, instead of carbon paper supported platinum, was used as anode material. The coulombic efficiency had been substantially improved from 1.68% to 42.49%, higher than other HPB-based MFCs previously reported. The SEM image proved the ability of XM02 strain to colonize on the anode surface. Power generation of MFCs could restore quickly when anodic medium was completely replaced with non-growth medium containing glucose. This suggested that the attached cells contributed to electricity production because planktonic cells had been removed during the medium replacement. This study proposed the mechanism of power generated from in situ oxidation of hydrogen produced by the XM02 strain biofilm.

  1. Recombination-induced suppression of cell division following P1-mediated generalized transduction in Klebsiella aerogenes.

    PubMed

    Bender, R A; Sambucetti, L C

    1983-01-01

    Klebsiella aerogenes recombinants resulting from bacteriophage P1-mediated generalized transduction failed to increase in number for approximately six generations after transduction. Nevertheless these recombinants continued to grow and became sensitive to penicillin after a transient resistance, suggesting that the cells were growing as long, non-dividing filaments. When filamentous cells were isolated from transduced cultures by gradient centrifugation, recombinants were 1000-fold more frequent among the filaments than among the normal-sized cells. The suppression of cell-division lasted for six generations whether markers near the origin (gln, ilv) or terminus (his, trp) of chromosome replication were used, despite a 50-fold difference in transduction frequencies for these markers. The suppression of cell division was a host response to recombination rather than to P1 invasion since cells lysogenized by P1 in these same experiments showed only a short (two generation) suppression of cell division. We speculate that the suppression of cell-division is an SOS response triggered by the degraded DNA not incorporated in the final recombinant. We demonstrate that both the filamentation and the transient penicillin resistance of recombinant cells can be exploited to enrich greatly for recombinants, raising transduction frequencies to as high as 10(-3).

  2. Recombinant expression of Laceyella sacchari thermitase in Lactococcus lactis.

    PubMed

    Jørgensen, Casper M; Madsen, Søren M; Vrang, Astrid; Hansen, Ole C; Johnsen, Mads G

    2013-12-01

    Thermitase (EC 3.4.21.66) is a thermostable endo-protease with the ability to convert various food relevant substrates into low-molecular weight peptides. A thermitase produced by Laceyella sacchari strain DSM43353 was found to have a mature amino acid sequence nearly identical to that of the original thermitase isolated from Thermoactinomyces vulgaris. The DSM43353 thermitase gene sequence contains a pro-peptide including parts of an I9 inhibitor motif. Expression of the thermitase gene in the Lactococcus lactis P170 expression system allowed secretion of stable thermitase in an auto-induced fermentation setup at 30°C. Thermitase accumulated in the culture supernatant during batch fermentations and was easily activated at 50°C or by prolonged dialysis. The activation step resulted in an almost complete degradation of endogenous L. lactis host proteins present in the supernatant. Mature activated product was stable at 50°C and functional at pH values between pH 6 and pH 11, suggesting that substrate hydrolysis can be performed over a broad range of pH values. The L. lactis based P170 expression system is a simple and safe system for obtaining food compatible thermitase in the range of 100 mg/L.

  3. Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

    PubMed

    Larsen, Nadja; Moslehi-Jenabian, Saloomeh; Werner, Birgit Brøsted; Jensen, Maiken Lund; Garrigues, Christel; Vogensen, Finn Kvist; Jespersen, Lene

    2016-06-01

    Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production. PMID:27015296

  4. Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

    PubMed

    Larsen, Nadja; Moslehi-Jenabian, Saloomeh; Werner, Birgit Brøsted; Jensen, Maiken Lund; Garrigues, Christel; Vogensen, Finn Kvist; Jespersen, Lene

    2016-06-01

    Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production.

  5. Enzymatic synthesis of L-tryptophan by Enterobacter aerogenes tryptophanase highly expressed in Escherichia coli, and some properties of the purified enzyme.

    PubMed

    Kawasaki, K; Yokota, A; Tomita, F

    1995-10-01

    We constructed two plasmids that have a strong tac promoter and a structural gene for tryptophanase of Enterobacter aerogenes SM-18 (pKT901EA) or Escherichia coli K-12 (pKT951EC). The tryptophanase activity of E. coli JM109 transformed with pKT901EA (JM109/pKT901EA) was inducible with isopropyl-beta-D-thiogalactopyranoside, and 3.6 times higher than that of E. aerogenes SM-18. Cells of JM109/pKT901EA induced for tryptophanase synthesized L-tryptophan from indole, ammonia, and pyruvate more efficiently than E. aerogenes SM-18. Although JM109/pKT951EC expressed a similar level of tryptophanase activity to that of JM109/pKT901EA, the synthesis of L-tryptophan by the cells of JM109/pKT951EC did not proceed well compared with JM109/pKT901EA. Tryptophanases from E. aerogenes and E. coli K-12 were purified, and their properties were investigated. The purified E. aerogenes tryptophanase showed higher stability against heat inactivation than E. coli tryptophanase.

  6. Complete genome sequence of Lactococcus lactis S0, an efficient producer of nisin.

    PubMed

    Zhao, Fangyuan; Ma, Hongchu; Lu, Ying; Teng, Kunling; Kang, Xusheng; Wang, Fangfang; Yang, Xiaopan; Zhong, Jin

    2015-03-20

    Lactococcus lactis S0 is a nisin Z-producing strain isolated from milk, and the nisin production of the strain can reach 4000 IU/ml under fermenting condition. Here, we present the complete genome sequence of L. lactis S0 which includes a single circular chromosome.

  7. Genome Sequence of a Lactococcus lactis Strain Isolated from Salmonid Intestinal Microbiota.

    PubMed

    Opazo, Rafael; Gajardo, Felipe; Ruiz, Mauricio; Romero, Jaime

    2016-01-01

    Lactococcus lactis is a common inhabitant of the intestinal microbiota of salmonids, especially those in aquaculture systems. Here, we present a genome sequence of a Lactococcus lactis strain isolated from the intestinal contents of rainbow trout reared in Chile. PMID:27563049

  8. Genome Sequence of a Lactococcus lactis Strain Isolated from Salmonid Intestinal Microbiota

    PubMed Central

    Opazo, Rafael; Gajardo, Felipe; Ruiz, Mauricio

    2016-01-01

    Lactococcus lactis is a common inhabitant of the intestinal microbiota of salmonids, especially those in aquaculture systems. Here, we present a genome sequence of a Lactococcus lactis strain isolated from the intestinal contents of rainbow trout reared in Chile. PMID:27563049

  9. Cloning and analysis of the pepV dipeptidase gene of Lactococcus lactis MG1363.

    PubMed

    Hellendoorn, M A; Franke-Fayard, B M; Mierau, I; Venema, G; Kok, J

    1997-06-01

    The gene pepV, encoding a dipeptidase from Lactococcus lactis subsp. cremoris MG1363, was identified in a genomic library in pUC19 in a peptidase-deficient Escherichia coli strain and subsequently sequenced. PepV of L. lactis is enzymatically active in E. coli and hydrolyzes a broad range of dipeptides but no tri-, tetra-, or larger oligopeptides. Northern (RNA) and primer extension analyses indicate that pepV is a monocistronic transcriptional unit starting 24 bases upstream of the AUG translational start codon. The dipeptidase of L. lactis was shown to be similar to the dipeptidase encoded by pepV of L. delbrueckii subsp. lactis, with 46% identity in the deduced amino acid sequences. A PepV-negative mutant of L. lactis was constructed by single-crossover recombination. Growth of the mutant strain in milk was significantly slower than that of the wild type, but the strains ultimately reached the same final cell densities.

  10. Encapsulated Lactococcus lactis with enhanced gastrointestinal survival for the development of folate enriched functional foods.

    PubMed

    Divya, Jayakumar Beena; Nampoothiri, Kesavan Madhavan

    2015-01-01

    Two lactic acid bacteria (LAB) isolated from cow's milk were identified as Lactococcus lactis strains and designated as L. lactis CM22 and L. lactis CM28. They were immobilised by co-encapsulation using alginate and mannitol and by hybrid entrapment with skim milk, glycerol, CaCO3 and alginate. The encapsulated cells survived better in simulated gastrointestinal conditions compared to the free cells. The percentage survival of probiotics encapsulated by hybrid entrapment method was 62.74% for L. lactis CM22 and 68% for L. lactis CM28. Studies to check their efficacy in fermentative fortification of skim milk and ice cream revealed an enhancement in folate level. PMID:25686721

  11. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10.

    PubMed

    Steidler, Lothar; Neirynck, Sabine; Huyghebaert, Nathalie; Snoeck, Veerle; Vermeire, An; Goddeeris, Bruno; Cox, Eric; Remon, Jean Paul; Remaut, Erik

    2003-07-01

    Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.

  12. Complete genome sequence of Lactococcus lactis IO-1, a lactic acid bacterium that utilizes xylose and produces high levels of L-lactic acid.

    PubMed

    Kato, Hiroaki; Shiwa, Yuh; Oshima, Kenshiro; Machii, Miki; Araya-Kojima, Tomoko; Zendo, Takeshi; Shimizu-Kadota, Mariko; Hattori, Masahira; Sonomoto, Kenji; Yoshikawa, Hirofumi

    2012-04-01

    We report the complete genome sequence of Lactococcus lactis IO-1 (= JCM7638). It is a nondairy lactic acid bacterium, produces nisin Z, ferments xylose, and produces predominantly L-lactic acid at high xylose concentrations. From ortholog analysis with other five L. lactis strains, IO-1 was identified as L. lactis subsp. lactis.

  13. Bioproduction of D-psicose from allitol with Enterobacter aerogenes IK7: a new frontier in rare ketose production.

    PubMed

    Gullapalli, Pushpakiran; Takata, Goro; Poonperm, Wayoon; Rao, Devendar; Morimoto, Kenji; Akimitsu, Kazuya; Tajima, Shigeyuki; Izumori, Ken

    2007-12-01

    D-psicose, a new alternative sweetener, was produced from allitol by microbial oxidation of the newly isolated strain Enterobacter aerogenes IK7. Cells grown in tryptic soy broth medium (TSB) supplemented with D-mannitol at 37 degrees C were found to have the best oxidation potential. The cells, owing to broad substrate specificity, oxidized various polyols (tetritol, pentitol, and hexitol) to corresponding rare ketoses. By a resting cell reaction, 10% of allitol was completely transformed to the product D-psicose, which thus becomes economically feasible for the mass production of D-psicose. Finally, the product was crystallized and confirmed to be D-psicose by analytical methods.

  14. Expression of NAD+-dependent formate dehydrogenase in Enterobacter aerogenes and its involvement in anaerobic metabolism and H2 production.

    PubMed

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Wu, Xi; Xing, Xin-Hui

    2009-10-01

    An expression system for NAD(+)-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H(2) yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H(2) production.

  15. BACT analysis: Are there cost effective air quality benefits from trees?

    SciTech Connect

    McPherson, E.G.; Simpson, J.R.; Scott, K.I.

    1996-12-31

    Trees absorb gaseous pollutants through leaf stomata and can bind or dissolve water soluble pollutants onto moist leaf surfaces. Tree canopies also intercept particulates and reduce local air temperatures. Urban trees may reduce ambient air ozone concentrations, either by direct absorption of ozone or other pollutants such as NO{sub 2}, or by reducing air temperatures, which reduces hydrocarbon emission and ozone formation rates. On the other hand, biogenic hydrocarbon emissions from trees may play a role in ozone formation. The role of trees in air quality has become coupled with concern over the costs and benefits of large-scale urban free planting programs. Air quality management districts provide pollution abatement credits to businesses and institutions by permitting the use of controls or processes, provided they are technically feasible and cost effective, based upon guidelines in Best Available Control Technology (BACT) manuals. Typically BACT analysis is applied to stationary sources, but the authors apply it here to determine if a large-scale urban tree planting can be a cost effective means to improve air quality.

  16. Characterization of Leuconostoc lactis strains from human sources.

    PubMed Central

    Barreau, C; Wagener, G

    1990-01-01

    An examination of 23 vancomycin-resistant, streptococcuslike isolates of clinical origin revealed a group of 14 to be related to Leuconostoc lactis (type strain, CIP 102422) on the basis of chemotaxonomic studies. These isolates were initially shown to be atypical by classical biochemical tests. However, they were characterized in particular by their polar lipid patterns by thin-layer chromatography and, additionally, by whole-cell protein patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is feasible, therefore, that common biochemical tests may continue to serve the purpose of routine identification, even though such isolates were formerly thought to be only of dairy origin. Images PMID:2394800

  17. Biohydrogen and Bioethanol Production from Biodiesel-Based Glycerol by Enterobacter aerogenes in a Continuous Stir Tank Reactor.

    PubMed

    Jitrwung, Rujira; Yargeau, Viviane

    2015-01-01

    Crude glycerol from the biodiesel manufacturing process is being produced in increasing quantities due to the expanding number of biodiesel plants. It has been previously shown that, in batch mode, semi-anaerobic fermentation of crude glycerol by Enterobacter aerogenes can produce biohydrogen and bioethanol simultaneously. The present study demonstrated the possible scaling-up of this process from small batches performed in small bottles to a 3.6-L continuous stir tank reactor (CSTR). Fresh feed rate, liquid recycling, pH, mixing speed, glycerol concentration, and waste recycling were optimized for biohydrogen and bioethanol production. Results confirmed that E. aerogenes uses small amounts of oxygen under semi-anaerobic conditions for growth before using oxygen from decomposable salts, mainly NH4NO3, under anaerobic condition to produce hydrogen and ethanol. The optimal conditions were determined to be 500 rpm, pH 6.4, 18.5 g/L crude glycerol (15 g/L glycerol) and 33% liquid recycling for a fresh feed rate of 0.44 mL/min. Using these optimized conditions, the process ran at a lower media cost than previous studies, was stable after 7 days without further inoculation and resulted in yields of 0.86 mol H2/mol glycerol and 0.75 mol ethanol/mole glycerol. PMID:25970750

  18. Biohydrogen and Bioethanol Production from Biodiesel-Based Glycerol by Enterobacter aerogenes in a Continuous Stir Tank Reactor

    PubMed Central

    Jitrwung, Rujira; Yargeau, Viviane

    2015-01-01

    Crude glycerol from the biodiesel manufacturing process is being produced in increasing quantities due to the expanding number of biodiesel plants. It has been previously shown that, in batch mode, semi-anaerobic fermentation of crude glycerol by Enterobacter aerogenes can produce biohydrogen and bioethanol simultaneously. The present study demonstrated the possible scaling-up of this process from small batches performed in small bottles to a 3.6-L continuous stir tank reactor (CSTR). Fresh feed rate, liquid recycling, pH, mixing speed, glycerol concentration, and waste recycling were optimized for biohydrogen and bioethanol production. Results confirmed that E. aerogenes uses small amounts of oxygen under semi-anaerobic conditions for growth before using oxygen from decomposable salts, mainly NH4NO3, under anaerobic condition to produce hydrogen and ethanol. The optimal conditions were determined to be 500 rpm, pH 6.4, 18.5 g/L crude glycerol (15 g/L glycerol) and 33% liquid recycling for a fresh feed rate of 0.44 mL/min. Using these optimized conditions, the process ran at a lower media cost than previous studies, was stable after 7 days without further inoculation and resulted in yields of 0.86 mol H2/mol glycerol and 0.75 mol ethanol/mole glycerol. PMID:25970750

  19. Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes.

    PubMed

    Martins, A; Spengler, G; Martins, M; Rodrigues, L; Viveiros, M; Davin-Regli, A; Chevalier, J; Couto, I; Pagès, J M; Amaral, L

    2010-10-01

    Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively.

  20. Effects of carbon source and Vitreoscilla hemoglobin (VHb) on the production of beta-galactosidase in Enterobacter aerogenes.

    PubMed

    Khleifat, Khaled M; Abboud, Muayad M; Al-Mustafa, Ahmed H; Al-Sharafa, Khalid Y

    2006-10-01

    At fixed concentration (0.5%), lactose and galactose acted as inducers while glucose and other tested carbon sugars showed repression effects on beta-galactosidase production in Enterobacter aerogenes strain. The expression of Vitreoscilla hemoglobin gene (vgb) in this bacterial strain managed to overcome the repression effects as well as improving the induction of beta-galactosidase formation by carbon sources. In parallel, the bacterial O(2) consumption was increased correspondingly to the vgb induction of beta-galactosidase synthesis. When Enterobacter aerogenes strains were grown at the incubation temperature 42 degrees C, about 5-fold higher enzyme productivity was obtained than with a similar incubation at 37 degrees C. The bacterial growth expressed as biomass yield had a different optimum temperature and was not influenced to the same extent by variations in the carbon sources. These data are discussed in terms of proposed enhancement in beta-galactosidase productivity by vgb expression as well as its significance to improve the technology of whey processing.

  1. Biohydrogen and polyhydroxyalkanoate co-production by Enterobacter aerogenes and Rhodobacter sphaeroides from Calophyllum inophyllum oil cake.

    PubMed

    Arumugam, A; Sandhya, M; Ponnusami, V

    2014-07-01

    The feasibility of coupled biohydrogen and polyhydroxyalkanoate production by Enterobacter aerogenes and Rhodobacter sphaeroides using Calophyllum inophyllum oil cake was studied under dark and photo fermentation conditions. The utilization of a non-edible acidic oil cake (C. inophyllum), and exploitation of a modified minimal salt media led to reduction in the cost of media. Cost of fermentation is reduced by implementation of alternate dark-photo fermentative periods and through the use of a co-culture consisting of a dark fermentative (E. aerogenes) and a photo fermentative (R. sphaeroides) bacterium. The biohydrogen and polyhydroxyalkanoate produced were 7.95 L H2/L media and 10.73 g/L media, respectively, under alternate dark and photo fermentation and were 3.23 L H2/L media and 5.6g/L media, respectively under complete dark fermentation. The characteristics of the oil cake and alternate dark (16 h) and photo (8h) fermentative conditions were found to be supportive in producing high biohydrogen and polyhydroxyalkanoate (PHA) yield.

  2. Biohydrogen and Bioethanol Production from Biodiesel-Based Glycerol by Enterobacter aerogenes in a Continuous Stir Tank Reactor.

    PubMed

    Jitrwung, Rujira; Yargeau, Viviane

    2015-05-11

    Crude glycerol from the biodiesel manufacturing process is being produced in increasing quantities due to the expanding number of biodiesel plants. It has been previously shown that, in batch mode, semi-anaerobic fermentation of crude glycerol by Enterobacter aerogenes can produce biohydrogen and bioethanol simultaneously. The present study demonstrated the possible scaling-up of this process from small batches performed in small bottles to a 3.6-L continuous stir tank reactor (CSTR). Fresh feed rate, liquid recycling, pH, mixing speed, glycerol concentration, and waste recycling were optimized for biohydrogen and bioethanol production. Results confirmed that E. aerogenes uses small amounts of oxygen under semi-anaerobic conditions for growth before using oxygen from decomposable salts, mainly NH4NO3, under anaerobic condition to produce hydrogen and ethanol. The optimal conditions were determined to be 500 rpm, pH 6.4, 18.5 g/L crude glycerol (15 g/L glycerol) and 33% liquid recycling for a fresh feed rate of 0.44 mL/min. Using these optimized conditions, the process ran at a lower media cost than previous studies, was stable after 7 days without further inoculation and resulted in yields of 0.86 mol H2/mol glycerol and 0.75 mol ethanol/mole glycerol.

  3. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    PubMed

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese.

  4. Oral Administration of Recombinant Lactococcus lactis Expressing the Cellulase Gene Increases Digestibility of Fiber in Geese.

    PubMed

    Zhou, Haizhu; Gao, Yunhang; Gao, Guang; Lou, Yujie

    2015-12-01

    Enhancing cellulose digestibility in animals is important for improving the utilization of forage, which can decrease the amount of food used in animal production. The aim of the present study was to achieve recombinant expression of the cellulase gene in Lactococcus lactis and evaluate the effects of oral administration of the recombinant L. lactis on fiber digestibility in geese. Cellulase (Cell) and green fluorescent protein (GFP) genes were cloned into a L. lactis expression vector (pNZ8149) to construct the recombinant expression plasmid (pNZ8149-GFP-Cell). Then, the recombinant expression plasmid was transformed into L. lactis (NZ3900) competent cells by electroporation to obtain recombinant L. lactis (pNZ8149-GFP-Cell/NZ3900) in which protein expression was induced by Nisin. Expression of GFP and Cell by the recombinant L. lactis was confirmed using SDS-PAGE, fluorescence detection, and Congo red assays. A feeding experiment showed that oral administration of pNZ8149-GFP-Cell/NZ3900 significantly increased the digestibility of dietary fiber in geese fed either a maize stalk diet or a rice chaff diet. Therefore, oral administration of recombinant L. lactis cells expressing the cellulase gene increases fiber digestibility in geese, offering a way to increase the utilization of dietary fiber in geese. PMID:26341925

  5. Sequencing and transcriptional analysis of the biosynthesis gene cluster of putrescine-producing Lactococcus lactis.

    PubMed

    Ladero, Victor; Rattray, Fergal P; Mayo, Baltasar; Martín, María Cruz; Fernández, María; Alvarez, Miguel A

    2011-09-01

    Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution.

  6. Electron microscopy of virulent phages for Streptococcus lactis.

    PubMed Central

    Tsaneva, K P

    1976-01-01

    Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern. Images PMID:817668

  7. Characterization of Lactococcus lactis isolates from bovine mastitis.

    PubMed

    Plumed-Ferrer, Carme; Uusikylä, Kaisa; Korhonen, Jenni; von Wright, Atte

    2013-12-27

    Lactococcus lactis is a widely used mesophilic dairy starter and has been included in the Qualified Presumption of Safety (QPS) list of the European Food Safety Authority. However, it is increasingly found as the cause of human or animal infections, such as bovine mastitis, probably due to the improvement of the identification of the infective microorganisms. Since there are some grounds to suspect that at least certain variants of L. lactis may cause animal or human diseases, it is important to properly identify the differences between the strains associated with infections and the safe starter strains. Bovine mastitis isolates and dairy starter strains were genotypically characterized and clustered by the 16S rRNA gene sequence and RAPD-PCR fingerprint patterns, and phenotypically characterized by their tolerance to different stress conditions typically found in the intestinal tract of mammals, the carbohydrate- and antibiotic resistance profile, as well as the in vitro adhesion capacity to udder epithelial cells. Genotypically, there were no differences between the mastitis isolates and the dairy starter strains. However, there were clear phenotypic distinctions between mastitis isolates and typical starter strains, the former showing an increased tolerance to temperature, lysozyme, bile salts, pH and antibiotics, as well as improved carbohydrate fermentation capacity, and in vitro adhesion to udder epithelial cells. Although these differences might not be considered as actual virulence factors, they improve the ability of the strain to survive in the body of homeothermic animals and, interestingly, are also typical properties associated with potential probiotic strains.

  8. Functional Expression of an Orchid Fragrance Gene in Lactococcus lactis

    PubMed Central

    Song, Adelene Ai Lian; Abdullah, Janna O.; Abdullah, Mohd Puad; Shafee, Norazizah; Rahim, Raha A.

    2012-01-01

    Vanda Mimi Palmer (VMP), an orchid hybrid of Vanda tesselata and Vanda Tan Chay Yan is a highly scented tropical orchid which blooms all year round. Previous studies revealed that VMP produces a variety of isoprenoid volatiles during daylight. Isoprenoids are well known to contribute significantly to the scent of most fragrant plants. They are a large group of secondary metabolites which may possess valuable characteristics such as flavor, fragrance and toxicity and are produced via two pathways, the mevalonate (MVA) pathway or/and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. In this study, a sesquiterpene synthase gene denoted VMPSTS, previously isolated from a floral cDNA library of VMP was cloned and expressed in Lactococcus lactis to characterize the functionality of the protein. L. lactis, a food grade bacterium which utilizes the mevalonate pathway for isoprenoid production was found to be a suitable host for the characterization of plant terpene synthases. Through recombinant expression of VMPSTS, it was revealed that VMPSTS produced multiple sesquiterpenes and germacrene D dominates its profile. PMID:22408409

  9. Failure of the MicroScan WalkAway system to detect heteroresistance to carbapenems in a patient with Enterobacter aerogenes bacteremia.

    PubMed

    Gordon, N C; Wareham, D W

    2009-09-01

    We report the failure of the automated MicroScan WalkAway system to detect carbapenem heteroresistance in Enterobacter aerogenes. Carbapenem resistance has become an increasing concern in recent years, and robust surveillance is required to prevent dissemination of resistant strains. Reliance on automated systems may delay the detection of emerging resistance.

  10. Isolation of Enterobacter aerogenes carrying blaTEM-1 and blaKPC-3 genes recovered from a hospital Intensive Care Unit.

    PubMed

    Pulcrano, Giovanna; Pignanelli, Salvatore; Vollaro, Adriana; Esposito, Matilde; Iula, Vita Dora; Roscetto, Emanuela; Soriano, Amata Amy; Catania, Maria Rosaria

    2016-06-01

    Enterobacter aerogenes has recently emerged as an important hospital pathogen. In this study, we showed the emergence of E. aerogenes isolates carrying the blaKPC gene in patients colonized by carbapenem-resistant Klebsiella pneumoniae strains. Two multiresistant E. aerogenes isolates were recovered from bronchial aspirates of two patients hospitalized in the Intensive Care Unit at the "Santa Maria della Scaletta" Hospital, Imola. The antimicrobial susceptibility test showed the high resistance to carbapenems and double-disk synergy test confirmed the phenotype of KPC and AmpC production. Other investigation revealed that ESBL and blaKPC genes were carried on the conjugative pKpQIL plasmid. This is a relevant report in Italy that describes a nosocomial infection due to the production of KPC beta-lactamases by an E. aerogenes isolate in patients previously colonized by K. pneumoniae carbapenem-resistant. In conclusion, it's necessary a continuous monitoring of multidrug-resistant strains for the detection of any KPC-producing bacteria that could expand the circulation of carbapenem-resistant pathogens.

  11. Antilisterial Activity of Nisin-Like Bacteriocin-Producing Lactococcus lactis subsp. lactis Isolated from Traditional Sardinian Dairy Products

    PubMed Central

    Cosentino, Sofia; Fadda, Maria Elisabetta; Deplano, Maura; Melis, Roberta; Pomata, Rita; Pisano, Maria Barbara

    2012-01-01

    With the aim of selecting LAB strains with antilisterial activity to be used as protective cultures to enhance the safety of dairy products, the antimicrobial properties of 117 Lactococcus lactis subsp. lactis isolated from artisanal Sardinian dairy products were evaluated, and six strains were found to produce bacteriocin-like substances. The capacity of these strains to antagonize Listeria monocytogenes during cocultivation in skimmed milk was evaluated, showing a reduction of L. monocytogenes counts of approximately 4 log units compared to the positive control after 24 h of incubation. In order for a strain to be used as bioprotective culture, it should be carefully evaluated for the presence of virulence factors, to determine what potential risks might be involved in its use. None of the strains tested was found to produce biogenic amines or to possess haemolytic activity. In addition, all strains were sensitive to clinically important antibiotics such as ampicillin, tetracycline, and vancomycin. Our results suggest that these bac+ strains could be potentially applied in cheese manufacturing to control the growth of L. monocytogenes. PMID:22536018

  12. An evaluation and partial characterization of a bacteriocin produced by Lactococcus lactis subsp lactis ST1 isolated from goat milk

    PubMed Central

    Taheri, Parinaz; Samadi², Nasrin; Ehsani, Mohammad Reza; Khoshayand, Mohammad Reza; Jamalifar, Hossein

    2012-01-01

    A bacteriocin-like inhibitory substance producing Lactococcus lactis subsp lactis strain, ST1, isolated from goat milk of Iranian origin and with broad spectrum of activity and desirable technical properties was used for evaluating some futures of bacteriocin inhibitory activity. Cell growth and bacteriocin production studies were carried out in MRS medium incubated statically under uncontrolled pH condition. The antibacterial activity presented a primary metabolite pattern and showed a rapid decrease at the stationary phase. Microaerobiosis and capnophily growth conditions resulted in higher bacteriocin production while aerobiosis showed negative effect on both cell growth and bacteriocin production. Bacteriocin production, on the other hand, was favored in MRS broth (pH; 6.5) inoculated with 0.1 ml l-1 fresh culture when incubation was carried out at 30 °C. This indicated that the conditions resulted in higher levels of growth were frequently favoring bacteriocin production by ST1 as well. Decrease in activity, at the stationary growth phase, was much pronounced in favored growth condition. Nutrient depletion, deferent effect of low pH on bacteriocin production and/or protein degradation seemed more responsible for this phenomenon. The study also provided further data on new method for bacteriocin release from the cell wall of producer. It was clearly shown that both heating and ultrasound shock for 5 min at pH 2 could increase bacteriocin activity significantly. The release was more pronounced in the presence of 0.5% Tween80. PMID:24031976

  13. L+-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84.

    PubMed

    Petrov, Kaloyan; Urshev, Zoltan; Petrova, Penka

    2008-06-01

    A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4.

  14. L+-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp. lactis B84.

    PubMed

    Petrov, Kaloyan; Urshev, Zoltan; Petrova, Penka

    2008-06-01

    A new Lactococcus lactis subsp. lactis B84, capable of utilizing starch as a sole carbon source and producing L(+)-lactate, was isolated from spontaneously fermented rye sourdough. Aiming at maximum lactic acid productivity, the components of the media and the cultivation conditions were varied. In MRS-starch medium (with absence of yeast and meat extracts), at 33 degrees C, agitation 200 rpm and pH 6.0 for 6 days complete starch hydrolysis occurred and 5.5 gl(-1) lactic acid were produced from 18 gl(-1) starch. The identification of strain B84 was based on genetic criteria. Amplified ribosomal DNA restriction analysis (ARDRA), PCR with species-specific primers and sequencing of the 16S rDNA proved its species affiliation. Four genes for enzymes, involved in starch degradation were detected in B84 genome: amyL, amyY, glgP and apu, coding cytoplasmic and extracellular alpha-amylases, glycogen phosphorylase and amylopullulanase, respectively. Reverse transcription PCR experiments showed that both genes, encoding alpha-amylases (amyL and amyY) were expressed into mRNAs, whereas apu and glgP were not. Amylase activity assay was performed at different pH and temperatures. The cell-bond amylase proved to be the key enzyme, involved in the starch hydrolysis with maximum activity at 45 degrees C and pH 5.4. PMID:18456109

  15. Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer.

    PubMed Central

    Mills, D A; Choi, C K; Dunny, G M; McKay, L L

    1994-01-01

    The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp. lactis ML3 plasmid pRS01 were localized by insertional mutagenesis. Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency. Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer. Tra3 corresponds closely to a region previously identified (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype. Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3. When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants. PMID:7811081

  16. Effect of selenium-enriched exopolysaccharide produced by Lactococcus lactis subsp. lactis on signaling molecules in mouse spleen lymphocytes.

    PubMed

    Liu, Lu; Pan, Daodong; Zeng, Xiaoqun; Li, Hua

    2013-10-01

    Selenium-enriched exopolysaccharides (Se-EPS) produced by Lactococcus lactis subsp. lactis can be used as a safe and effective selenium (Se) supplement. Analysis of the Se content and monosaccharide components of Se-EPS demonstrated that it consisted of mannose, fucose, ribose, glucose, galactose, and arabinose with a molar ratio of 5.48 : 0.39 : 9.77 : 4.03 : 1.00 : 1.92, and an Se content of 183.263 μg g(-1). The differing effects of Se-EPS and EPS on calcium channels and certain key secondary messengers in spleen lymphocytes were examined and compared. Results showed that low-dose Se-EPS, but not EPS, increased the intracellular calcium ([Ca(2+)]i) levels of mouse spleen lymphocytes. Se-EPS also increased the expression and phosphorylation of Ca(2+)-calmodulin-dependent kinase (CaMK) II in lymphocytes. In addition, increased intracellular Ca(2+) levels were also found in the cells with blocked Ca(2+) channels. We speculated that Se-EPS enhanced the activation and proliferation of lymphocytes through calcium signaling by drawing from extracellular and intracellular stores. Low-dose Se-EPS also enhanced NO production, cAMP levels and PKA activity. We speculated that low-dose Se-EPS may activate certain pathways, including the calcium channel, NO, cAMP, and PKA related pathways.

  17. Antilisterial activity of nisin-like bacteriocin-producing Lactococcus lactis subsp. lactis isolated from traditional Sardinian dairy products.

    PubMed

    Cosentino, Sofia; Fadda, Maria Elisabetta; Deplano, Maura; Melis, Roberta; Pomata, Rita; Pisano, Maria Barbara

    2012-01-01

    With the aim of selecting LAB strains with antilisterial activity to be used as protective cultures to enhance the safety of dairy products, the antimicrobial properties of 117 Lactococcus lactis subsp. lactis isolated from artisanal Sardinian dairy products were evaluated, and six strains were found to produce bacteriocin-like substances. The capacity of these strains to antagonize Listeria monocytogenes during cocultivation in skimmed milk was evaluated, showing a reduction of L. monocytogenes counts of approximately 4 log units compared to the positive control after 24 h of incubation. In order for a strain to be used as bioprotective culture, it should be carefully evaluated for the presence of virulence factors, to determine what potential risks might be involved in its use. None of the strains tested was found to produce biogenic amines or to possess haemolytic activity. In addition, all strains were sensitive to clinically important antibiotics such as ampicillin, tetracycline, and vancomycin. Our results suggest that these bac+ strains could be potentially applied in cheese manufacturing to control the growth of L. monocytogenes. PMID:22536018

  18. Riboflavin Production in Lactococcus lactis: Potential for In Situ Production of Vitamin-Enriched Foods

    PubMed Central

    Burgess, Catherine; O'Connell-Motherway, Mary; Sybesma, Wilbert; Hugenholtz, Jeroen; van Sinderen, Douwe

    2004-01-01

    This study describes the genetic analysis of the riboflavin (vitamin B2) biosynthetic (rib) operon in the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NZ9000. Functional analysis of the genes of the L. lactis rib operon was performed by using complementation studies, as well as by deletion analysis. In addition, gene-specific genetic engineering was used to examine which genes of the rib operon need to be overexpressed in order to effect riboflavin overproduction. Transcriptional regulation of the L. lactis riboflavin biosynthetic process was investigated by using Northern hybridization and primer extension, as well as the analysis of roseoflavin-induced riboflavin-overproducing L. lactis isolates. The latter analysis revealed the presence of both nucleotide replacements and deletions in the regulatory region of the rib operon. The results presented here are an important step toward the development of fermented foods containing increased levels of riboflavin, produced in situ, thus negating the need for vitamin fortification. PMID:15466513

  19. Comparative genomics of Bifidobacterium animalis subsp. lactis reveals a strict monophyletic bifidobacterial taxon.

    PubMed

    Milani, Christian; Duranti, Sabrina; Lugli, Gabriele Andrea; Bottacini, Francesca; Strati, Francesco; Arioli, Stefania; Foroni, Elena; Turroni, Francesca; van Sinderen, Douwe; Ventura, Marco

    2013-07-01

    Strains of Bifidobacterium animalis subsp. lactis are extensively exploited by the food industry as health-promoting bacteria, although the genetic variability of members belonging to this taxon has so far not received much scientific attention. In this article, we describe the complete genetic makeup of the B. animalis subsp. lactis Bl12 genome and discuss the genetic relatedness of this strain with other sequenced strains belonging to this taxon. Moreover, a detailed comparative genomic analysis of B. animalis subsp. lactis genomes was performed, which revealed a closely related and isogenic nature of all currently available B. animalis subsp. lactis strains, thus strongly suggesting a closed pan-genome structure of this bacterial group. PMID:23645200

  20. Lactococcus lactis metabolism and gene expression during growth on plant tissues.

    PubMed

    Golomb, Benjamin L; Marco, Maria L

    2015-01-01

    Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations.

  1. Fate of Lactococcus lactis starter cultures during late ripening in cheese models.

    PubMed

    Ruggirello, Marianna; Cocolin, Luca; Dolci, Paola

    2016-10-01

    The presence of Lactococcus lactis, commonly employed as starter culture, was, recently, highlighted and investigated during late cheese ripening. Thus, the main goal of the present study was to assess the persistence and viability of this microorganism throughout manufacturing and ripening of model cheeses. Eight commercial starters, constituted of L. lactis subsp. lactis and L. lactis subsp. cremoris, were inoculated in pasteurized milk in order to manufacture miniature cheeses, ripened for six months. Samples were analysed at different steps (milk after inoculum, curd after cutting, curd after pressing and draining, cheese immediately after salting and cheese at 7, 15, 30, 60, 90, 120, 150 and 180 days of ripening) and submitted to both culture-dependent (traditional plating on M17) and -independent analysis (reverse transcription-quantitative PCR). On the basis of direct RNA analysis, L. lactis populations were detected in all miniature cheeses up to the sixth month of ripening, confirming the presence of viable cells during the whole ripening process, including late stages. Noteworthy, L. lactis was detected by RT-qPCR in cheese samples also when traditional plating failed to indicate its presence. This discrepancy could be explain with the fact that lactococci, during ripening process, enter in a stressed physiological state (viable not culturable, VNC), which might cause their inability to grow on synthetic medium despite their viability in cheese matrix. Preliminary results obtained by "resuscitation" assays corroborated this hypothesis and 2.5% glucose enrichment was effective to recover L. lactis cells in VNC state. The capability of L. lactis to persist in late ripening, and the presence of VNC cells which are known to shift their catabolism to peptides and amino acids consumption, suggests a possible technological role of this microorganism in cheese ripening with a possible impact on flavour formation. PMID:27375251

  2. Lactococcus lactis Metabolism and Gene Expression during Growth on Plant Tissues

    PubMed Central

    Golomb, Benjamin L.

    2014-01-01

    Lactic acid bacteria have been isolated from living, harvested, and fermented plant materials; however, the adaptations these bacteria possess for growth on plant tissues are largely unknown. In this study, we investigated plant habitat-specific traits of Lactococcus lactis during growth in an Arabidopsis thaliana leaf tissue lysate (ATL). L. lactis KF147, a strain originally isolated from plants, exhibited a higher growth rate and reached 7.9-fold-greater cell densities during growth in ATL than the dairy-associated strain L. lactis IL1403. Transcriptome profiling (RNA-seq) of KF147 identified 853 induced and 264 repressed genes during growth in ATL compared to that in GM17 laboratory culture medium. Genes induced in ATL included those involved in the arginine deiminase pathway and a total of 140 carbohydrate transport and metabolism genes, many of which are involved in xylose, arabinose, cellobiose, and hemicellulose metabolism. The induction of those genes corresponded with L. lactis KF147 nutrient consumption and production of metabolic end products in ATL as measured by gas chromatography-time of flight mass spectrometry (GC-TOF/MS) untargeted metabolomic profiling. To assess the importance of specific plant-inducible genes for L. lactis growth in ATL, xylose metabolism was targeted for gene knockout mutagenesis. Wild-type L. lactis strain KF147 but not an xylA deletion mutant was able to grow using xylose as the sole carbon source. However, both strains grew to similarly high levels in ATL, indicating redundancy in L. lactis carbohydrate metabolism on plant tissues. These findings show that certain strains of L. lactis are well adapted for growth on plants and possess specific traits relevant for plant-based food, fuel, and feed fermentations. PMID:25384484

  3. Heterologous expression of Aspergillus terreus fructosyltransferase in Kluyveromyces lactis.

    PubMed

    Spohner, Sebastian C; Czermak, Peter

    2016-06-25

    Fructo-oligosaccharides are prebiotic and hypocaloric sweeteners that are usually extracted from chicory. They can also be produced from sucrose using fructosyltransferases, but the only commercial enzyme suitable for this purpose is Pectinex Ultra, which is produced with Aspergillus aculeatus. Here we used the yeast Kluyveromyces lactis to express a secreted recombinant fructosyltransferase from the inulin-producing fungus Aspergillus terreus. A synthetic codon-optimised version of the putative β-fructofuranosidase ATEG 04996 (XP 001214174.1) from A. terreus NIH2624 was secreted as a functional protein into the extracellular medium. At 60°C, the purified A. terreus enzyme generated the same pattern of oligosaccharides as Pectinex Ultra, but at lower temperatures it also produced oligomers with up to seven units. We achieved activities of up to 986.4U/mL in high-level expression experiments, which is better than previous reports of optimised Aspergillus spp. fermentations. PMID:27084521

  4. Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice▿

    PubMed Central

    Medina, Marcela; Villena, Julio; Vintiñi, Elisa; Hebert, Elvira María; Raya, Raúl; Alvarez, Susana

    2008-01-01

    Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages. PMID:18390997

  5. Bifidobacterium lactis 420 and fish oil enhance intestinal epithelial integrity in Caco-2 cells.

    PubMed

    Mokkala, Kati; Laitinen, Kirsi; Röytiö, Henna

    2016-03-01

    Increased intestinal permeability is a predisposing factor for low-grade inflammation-associated conditions, including obesity and type 2 diabetes. Dietary components may influence intestinal barrier integrity. We hypothesized that the dietary supplements Bifidobacterium lactis 420, Lactobacillus rhamnosus HN001, and fish oil have beneficial impacts on intestinal barrier integrity. In addition, we hypothesized that the coadministration of these components results in synergistic benefits to the integrity of the intestinal barrier. To study this, we investigated the impact of cell-free culture supernatant from dietary supplements B lactis 420 and L rhamnosus HN001, and fish oil, separately and in combination, on intestinal permeability in a CaCo-2 cell model. Administered separately, both B lactis 420 supernatant and fish oil significantly increased the integrity of the intestinal epithelial barrier, as determined by an increase in transepithelial electrical resistance (TEER), whereas L rhamnosus did not. The TEER increase with B lactis 420 was dose dependent. Interestingly, a combination of B lactis 420 supernatant and fish oil negated the increase in TEER of the single components. mRNA expression of tight junction proteins, measured by real-time quantitative polymerase chain reaction, was not altered, but the mRNA expression of myosin light chain kinase increased after fish oil treatment. To conclude, single dietary components, namely, B lactis 420 and fish oil, induced beneficial effects on intestinal barrier integrity in vitro, whereas a combination of 2 beneficial test compounds resulted in a null effect. PMID:26923511

  6. Structure of wild-type and mutant repressors and of the control region of the rbt operon of Klebsiella aerogenes.

    PubMed

    Wu, J; Anderton-Loviny, T; Smith, C A; Hartley, B S

    1985-05-01

    Pentitol metabolism in Klebsiella aerogenes is encoded by continuous ribitol (rbt) and D-arabitol (dal) operons transcribed in bipolar fashion and sandwiched between long stretches of homologous DNA. The operons are separated by a central control region (2.2 kb) which encodes both the repressors and all the control sequences. The rbt repressor (270 amino acids) shows homology to the Escherichia coli lac repressor and other DNA-binding proteins. It is transcribed from the strand opposite the rbt operon and the intervening control region (254-bp) contains features which reflect the complex regulation. A rbt-constitutive mutant strain used in previous studies of experimental enzyme evolution encodes a truncated rbt-peptide of 133 residues due to a frameshift mutation. PMID:3891331

  7. Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria

    PubMed Central

    2010-01-01

    Background We studied the beta-lactamases of an E. aerogenes isolate recovered from the blood of a two-year-old patient. The isolate demonstrated a disk-diffusion phenotype typical for an AmpC-ESBL co-producer. Methods Microbiology studies were performed according to standard protocols. The resistance gene was identified by transconjugation and cloning experiments. Results By transconjugation only a narrow spectrum beta-lactamase (TEM-1) encoded on a small plasmid was transmitted. The ESBL was cloned and expressed in an E. coli host. Sequence analysis of the recombinant plasmid revealed blaSHV-12 associated to the insertion sequence, IS26. Conclusion This is the first study demonstrated the occurrence of SHV-12 in Nigeria. PMID:20067633

  8. Genome shuffling of Lactococcus lactis subspecies lactis YF11 for improving nisin Z production and comparative analysis.

    PubMed

    Zhang, Y F; Liu, S Y; Du, Y H; Feng, W J; Liu, J H; Qiao, J J

    2014-05-01

    Nisin has been widely used in the food industry as a safe and natural preservative to increase the shelf time of many foods. In this study, genome shuffling was applied to improve nisin Z production of Lactococcus lactis ssp. lactis YF11 (YF11) via recursive protoplast fusion. Ultraviolet irradiation and diethyl sulfate mutagenesis were used to generate parental strains for genome shuffling. After 4 rounds of genome shuffling, the best-performing strain F44 was obtained, which showed dramatic improvements in tolerance to both glucose (ranging from 8 to 15% (wt/vol) and nisin (ranging from 5,000 to 14,000 IU/mL). Fed-batch fermentation showed that the nisin titer of F44 was up to 4,023 IU/mL, which was 2.4 times that of the starting strain YF11. Field emission scanning electron microscope micrographs of YF11 and F44 revealed the apparent differences in cell morphology. Whereas YF11 displayed long and thin cell morphology, F44 cells were short and thick and with a raised surface in the middle of the cell. With the increasing glucose and nisin content in the medium, cells of both YF11 and F44 tended to become shrunken; however, alterations in YF11 cells were more pronounced than those of F44 cells, especially when cultured in tolerance medium containing both nisin and glucose. Nuclear magnetic resonance analysis demonstrated that the structure of nisin from YF11 and F44 was the same. Expression profiling of nisin synthesis related genes by real-time quantitative PCR showed that the transcription level of nisin structural gene nisZ and immunity gene nisI of F44 was 48 and 130% higher than that of the starting strain YF11, respectively. These results could provide valuable insights into the molecular basis underlying the nisin overproduction mechanism in L. lactis, thus facilitating the future construction of industrial strains for nisin production. PMID:24612797

  9. Genome shuffling of Lactococcus lactis subspecies lactis YF11 for improving nisin Z production and comparative analysis.

    PubMed

    Zhang, Y F; Liu, S Y; Du, Y H; Feng, W J; Liu, J H; Qiao, J J

    2014-05-01

    Nisin has been widely used in the food industry as a safe and natural preservative to increase the shelf time of many foods. In this study, genome shuffling was applied to improve nisin Z production of Lactococcus lactis ssp. lactis YF11 (YF11) via recursive protoplast fusion. Ultraviolet irradiation and diethyl sulfate mutagenesis were used to generate parental strains for genome shuffling. After 4 rounds of genome shuffling, the best-performing strain F44 was obtained, which showed dramatic improvements in tolerance to both glucose (ranging from 8 to 15% (wt/vol) and nisin (ranging from 5,000 to 14,000 IU/mL). Fed-batch fermentation showed that the nisin titer of F44 was up to 4,023 IU/mL, which was 2.4 times that of the starting strain YF11. Field emission scanning electron microscope micrographs of YF11 and F44 revealed the apparent differences in cell morphology. Whereas YF11 displayed long and thin cell morphology, F44 cells were short and thick and with a raised surface in the middle of the cell. With the increasing glucose and nisin content in the medium, cells of both YF11 and F44 tended to become shrunken; however, alterations in YF11 cells were more pronounced than those of F44 cells, especially when cultured in tolerance medium containing both nisin and glucose. Nuclear magnetic resonance analysis demonstrated that the structure of nisin from YF11 and F44 was the same. Expression profiling of nisin synthesis related genes by real-time quantitative PCR showed that the transcription level of nisin structural gene nisZ and immunity gene nisI of F44 was 48 and 130% higher than that of the starting strain YF11, respectively. These results could provide valuable insights into the molecular basis underlying the nisin overproduction mechanism in L. lactis, thus facilitating the future construction of industrial strains for nisin production.

  10. Protective effects of heat-killed Lactococcus lactis subsp. lactis BF3, isolated from the intestine of chum salmon, in a murine model of DSS-induced inflammatory bowel disease.

    PubMed

    Nakata, Toru; Hirano, Shino; Yokota, Yasushi; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi; Eto, Tadashi; Kato, Michiko

    2016-01-01

    Oxidative stress is considered an etiological factor responsible for several symptoms of inflammatory bowel disease (IBD). In vitro anti-inflammatory activities of heat-killed Lactococcus lactis subsp. lactis BF3 have been reported. In this study, the anti-inflammatory effect of these cells was examined using a dextran sodium sulphate (DSS)-induced murine IBD model. Administration of heat-killed L. lactis BF3 via drinking water suppressed the IBD symptoms, such as shortening of colon length, damage to the colon mucosa as observed under the microscope, and spleen enlargement. This result suggests that heat-killed L. lactis BF3 has the potential to treat IBD. PMID:27508115

  11. Protective effects of heat-killed Lactococcus lactis subsp. lactis BF3, isolated from the intestine of chum salmon, in a murine model of DSS-induced inflammatory bowel disease

    PubMed Central

    NAKATA, Toru; HIRANO, Shino; YOKOTA, Yasushi; TAKAHASHI, Hajime; KIMURA, Bon; KUDA, Takashi; ETO, Tadashi; KATO, Michiko

    2016-01-01

    Oxidative stress is considered an etiological factor responsible for several symptoms of inflammatory bowel disease (IBD). In vitro anti-inflammatory activities of heat-killed Lactococcus lactis subsp. lactis BF3 have been reported. In this study, the anti-inflammatory effect of these cells was examined using a dextran sodium sulphate (DSS)-induced murine IBD model. Administration of heat-killed L. lactis BF3 via drinking water suppressed the IBD symptoms, such as shortening of colon length, damage to the colon mucosa as observed under the microscope, and spleen enlargement. This result suggests that heat-killed L. lactis BF3 has the potential to treat IBD. PMID:27508115

  12. Epidemiological study of an outbreak due to multidrug-resistant Enterobacter aerogenes in a medical intensive care unit.

    PubMed Central

    Arpin, C; Coze, C; Rogues, A M; Gachie, J P; Bebear, C; Quentin, C

    1996-01-01

    In 1993, 63 isolates of Enterobacter aerogenes were collected from 41 patients in a medical intensive care unit (ICU). During the same period, only 46 isolates from 32 patients were collected in the rest of the hospital. All isolates were analyzed by antibiotic resistance phenotype, and 77 representative isolates were differentiated by plasmid restriction analysis, ribotyping, and arbitrarily primed (AP)-PCR. The extended-spectrum beta-lactamases produced by 22 strains were characterized by determination of their isoelectric points and by hybridization of plasmid DNA with specific probes. The isolates were divided into 25 antibiotic resistance phenotypes, either susceptible (group I) or resistant (group II) to aminoglycosides, and exhibited three phenotypes of resistance to beta-lactams: chromosomally derepressed cephalosporinase alone or associated with either extended-spectrum beta-lactamases (mainly of the SHV-4 type) or imipenem resistance. The results of the tests divided the 77 representative isolates (group I, n = 21; group II, n = 56) into 15 plasmid profiles, 14 ribotypes, and 15 AP-PCR patterns. Although the resistant isolates (group II) exhibited different plasmid profiles, ribotyping and AP-PCR analysis demonstrated an identical chromosomal pattern, indicating an epidemiological relatedness. They were mainly found in the medical ICU and occasionally in other units. The susceptible strains (group I) had various and distinct markers and were mainly isolated in units other than the medical ICU. In conclusion, the presence of a nosocomial outbreak in an ICU and the spread of a multidrug-resistant epidemic strain throughout the hospital was confirmed. Ribotyping and AP-PCR represent discriminatory tools for the investigation of nosocomial outbreaks caused by E. aerogenes. PMID:8862578

  13. Vibrio furnissii (formerly aerogenic biogroup of Vibrio fluvialis), a new species isolated from human feces and the environment.

    PubMed

    Brenner, D J; Hickman-Brenner, F W; Lee, J V; Steigerwalt, A G; Fanning, G R; Hollis, D G; Farmer, J J; Weaver, R E; Joseph, S W; Seidler, R J

    1983-10-01

    Strains formerly classified as the aerogenic (gas-producing) biogroup of Vibrio fluvialis were shown by DNA relatedness to be a separate species. The species was named Vibrio furnissii sp. nov. (type strain ATCC 35016 = CDC B3215). Three strains of V. furnissii were 79% or more related to the type strain of V. furnissii and about 50% related to the type strain of V. fluvialis. V. fluvialis strains were 40 to 64% related to the type strain of V. furnissii. Divergence in related sequences was only 0.0 to 1.5% among strains of V. furnissii and among strains of V. fluvialis but was 5.0 to 8.0% in interspecific reactions between V. fluvialis and V. furnissii. V. furnissii was aerogenic (produced gas from the fermentation of carbohydrates), whereas V. fluvialis was anaerogenic (did not produce gas from the fermentation of carbohydrates). Another test of some help in differentiating the two species was fermentation of L-rhamnose (57% positive for V. furnissii and negative for V. fluvialis). In addition to the reactions above, V. furnissii is distinguished from other salt-requiring vibrios on the basis of its positive reactions in tests for Møller L-arginine, L-arabinose, maltose, and D-mannitol and its negative reactions for Møller L-lysine and L-ornithine, lactose, and Voges-Proskauer. V. furnissii has been isolated from patients with acute gastroenteritis in at least two outbreaks of food poisoning; its role as a cause of diarrhea needs further study.

  14. Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

    PubMed Central

    Duwat, P; Ehrlich, S D; Gruss, A

    1992-01-01

    Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair. PMID:1514816

  15. Application of the ligase chain reaction to the detection of nisinA and nisinZ genes in Lactococcus lactis ssp. lactis.

    PubMed

    Ward, L J; Brown, J C; Davey, G P

    1994-03-15

    This paper reports on the application of the ligase chain reaction (LCR) to the specific detection of variants of the nisin structural gene (nisinA and nisinZ) in nisin producing strains of Lactococcus lactis ssp lactis. The LCR assay was used to screen nisin producing strains to determine which form of the nisin structural gene they contained. This method of differentiating the nisin structural gene variants provides a useful alternative to the only other available genetic differentiation, that of sequencing the gene.

  16. Evaluation of Lactococcus lactis Isolates from Nondairy Sources with Potential Dairy Applications Reveals Extensive Phenotype-Genotype Disparity and Implications for a Revised Species

    PubMed Central

    Cavanagh, Daniel; Casey, Aidan; Altermann, Eric; Cotter, Paul D.; Fitzgerald, Gerald F.

    2015-01-01

    Lactococcus lactis is predominantly associated with dairy fermentations, but evidence suggests that the domesticated organism originated from a plant niche. L. lactis possesses an unusual taxonomic structure whereby strain phenotypes and genotypes often do not correlate, which in turn has led to confusion in L. lactis classification. A bank of L. lactis strains was isolated from various nondairy niches (grass, vegetables, and bovine rumen) and was further characterized on the basis of key technological traits, including growth in milk and key enzyme activities. Phenotypic analysis revealed all strains from nondairy sources to possess an L. lactis subsp. lactis phenotype (lactis phenotype); however, seven of these strains possessed an L. lactis subsp. cremoris genotype (cremoris genotype), determined by two separate PCR assays. Multilocus sequence typing (MLST) showed that strains with lactis and cremoris genotypes clustered together regardless of habitat, but it highlighted the increased diversity that exists among “wild” strains. Calculation of average nucleotide identity (ANI) and tetranucleotide frequency correlation coefficients (TETRA), using the JSpecies software tool, revealed that L. lactis subsp. cremoris and L. lactis subsp. lactis differ in ANI values by ∼14%, below the threshold set for species circumscription. Further analysis of strain TIFN3 and strains from nonindustrial backgrounds revealed TETRA values of <0.99 in addition to ANI values of <95%, implicating that these two groups are separate species. These findings suggest the requirement for a revision of L. lactis taxonomy. PMID:25841018

  17. Immune responses elicited in mice with recombinant Lactococcus lactis expressing F4 fimbrial adhesin FaeG by oral immunization.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2010-08-01

    Enterotoxigenic Escherichia coli (ETEC) is a major pathogenic agent causing piglet diarrhea. The major subunit and adhesin FaeG of F4(+) ETEC is an important virulence factor with strong immunogenicity. To determine whether Lactococcus lactis (L. lactis) could effectively deliver FaeG to the mucosal immune system, recombinant L. lactis expressing FaeG was constructed, and immune responses in mice following oral route delivery of recombinant L. lactis were explored. The production of FaeG expressed in L. lactis was up to approximately 10% of soluble whole-cell proteins, and recombinant FaeG (rFaeG) possessed good immunoreactivity by Western blot analysis. Oral immunization with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses in the mice. In addition, high dose recombinant L. lactis or co-administration of high dose recombinant L. lactis with CTB enhanced the immune responses. These results suggested that L. lactis expressing FaeG was a promising candidate vaccine against ETEC. PMID:20532816

  18. Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

    PubMed Central

    Crow, Vaughan L.

    1990-01-01

    Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol. PMID:16348209

  19. Profil clinique et bactériologique des infections néonatales bactériennes à l'Hôpital Laquintinie de Douala, Cameroun

    PubMed Central

    Kemeze, Sandrine; Moudze, Béatrice; Chiabi, Andreas; Eposse, Charlotte; Kaya, Alexis; Mbangue, Madeleine; Guifo, Odette; Kago, Innocent

    2016-01-01

    Introduction L'Organisation Mondiale de la Santé a estimé la survenue globale de décès néonatal à 2,8 millions en 2015, dont 47,6% étaient dues aux infections. Ces infections peuvent survenir chez un nouveau-né de 0 à un mois de vie, pouvant aller jusqu’à 3 mois. Méthodes C'est une étude prospective allant du 1er mars au 30 juin 2015 au Service de néonatologie de l'Hôpital Laquintinie de Douala. Etaient inclus tout nouveau-né symptomatique avec ou sans critère anamnestique et tout nouveau-né asymptomatique, présentant au moins un risque infectieux et ayant au moins une culture positive ou une anomalie de la numération formule sanguine ou une protéine C réactive positive. Résultats Des 310 nouveau-nés admis, 300 ont été retenus pour infection néonatale, soit une incidence de 96,8%. Nous avons réalisé 104 cultures dont 25 positives, soit une incidence de l'infection néonatale confirmée de 24%. Les facteurs associés à l'infection étaient la prématurité inexpliquée <35 semaines d'aménorrhée(45,1%) et la réanimation néonatale (34,8%). La fièvre (56%) et les troubles neurologiques (48,8%) étaient les manifestations cliniques les plus fréquentes. Les Gram négatifs étaient les germes les plus fréquents (56%). L'imipenème (95%) et l'amikacine (66,7%) étaient les antibiotiques les plusefficaces. L’évolution était favorable dans 66,4% des cas et le taux de décès était de 33,6%. Conclusion Cette étude révèle une forte prévalence de l'infection dans cet hôpital. L’écologie bactérienne est dominée par les Gram négatifs, on note une importante résistance aux antibiotiques usuels et une mortalité assez élevée. PMID:27222688

  20. BactPepDB: a database of predicted peptides from a exhaustive survey of complete prokaryote genomes

    PubMed Central

    Rey, Julien; Deschavanne, Patrick; Tuffery, Pierre

    2014-01-01

    With the recent progress in complete genome sequencing, mining the increasing amount of genomic information available should in theory provide the means to discover new classes of peptides. However, annotation pipelines often do not consider small reading frames likely to be expressed. BactPepDB, available online at http://bactpepdb.rpbs.univ-paris-diderot.fr, is a database that aims at providing an exhaustive re-annotation of all complete prokaryotic genomes—chromosomal and plasmid DNA—available in RefSeq for coding sequences ranging between 10 and 80 amino acids. The identified peptides are classified as (i) previously identified in RefSeq, (ii) entity-overlapping (intragenic) or intergenic, and (iii) potential pseudogenes—intergenic sequences corresponding to a portion of a previously annotated larger gene. Additional information is related to homologs within order, predicted signal sequence, transmembrane segments, disulfide bonds, secondary structure, and the existence of a related 3D structure in the Protein Databank. As a result, BactPepDB provides insights about candidate peptides, and provides information about their conservation, together with some of their expected biological/structural features. The BactPepDB interface allows to search for candidate peptides in the database, or to search for peptides similar to a query, according to the multiple properties predicted or related to genomic localization. Database URL: http://www.yeastgenome.org/ PMID:25377257

  1. Lactose metabolism in Streptococcus lactis: studies with a mutant lacking glucokinase and mannose-phosphotransferase activities

    SciTech Connect

    Thompson, J.; Chassy, B.M.; Egan, W.

    1985-04-01

    A mutant of Streptococcus lactis 133 has been isolated that lacks both glucokinase and phosphoenolpyruvate-dependent mannose- phosphotransferase (mannose-PTS) activities. The double mutant S. lactis 133 mannose-PTSd GK- is unable to utilize either exogenously supplied or intracellularly generated glucose for growth. Fluorographic analyses of metabolites formed during the metabolism of (/sup 14/C)lactose labeled specifically in the glucose or galactosyl moiety established that the cells were unable to phosphorylate intracellular glucose. However, cells of S. lactis 133 mannose-PTSd GK- readily metabolized intracellular glucose 6-phosphate, and the growth rates and cell yield of the mutant and parental strains on sucrose were the same. During growth on lactose, S. lactis 133 mannose-PTSd GK- fermented only the galactose moiety of the disaccharide, and 1 mol of glucose was generated per mol of lactose consumed. For an equivalent concentration of lactose, the cell yield of the mutant was 50% that of the wild type. The specific rate of lactose utilization by growing cells of S. lactis 133 mannose-PTSd GK- was ca. 50% greater than that of the wild type, but the cell doubling times were 70 and 47 min, respectively. High-resolution /sup 31/P nuclear magnetic resonance studies of lactose transport by starved cells of S. lactis 133 and S. lactis 133 mannose-PTSd GK- showed that the latter cells contained elevated lactose-PTS activity. Throughout exponential growth on lactose, the mutant maintained an intracellular steady-state glucose concentration of 100 mM.

  2. A theoretical evidence for cooperative enhancement in aerogen-bonding interactions: Open-chain clusters of KrOF2 and XeOF2

    NASA Astrophysics Data System (ADS)

    Esrafili, Mehdi D.; Vessally, Esmail

    2016-10-01

    The cooperativity of aerogen-bonding interactions is studied in open-chain (KrOF2)n=2-6 and (XeOF2)n=2-6 clusters. The formation mechanism and bonding properties of these clusters are investigated by means of molecular electrostatic potentials, natural bond orbital and noncovalent interaction index analyses. The small variation of average interaction energy from the pentamer to hexamer cluster reveals that cooperativity effects are almost completely saturated in the larger clusters. The cooperative effects in the clusters also make an increase in the average 83Kr or 129Xe chemical shielding isotropies and total spin-spin coupling constants across the aerogen-bonding.

  3. Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines.

    PubMed

    Haghshenas, Babak; Abdullah, Norhafizah; Nami, Yousef; Radiah, Dayang; Rosli, Rozita; Khosroushahi, Ahmad Yari

    2014-12-01

    Lactobacillus and Lactococcus strains isolated from food products can be introduced as probiotics because of their health-promoting characteristics and non-pathogenic nature. This study aims to perform the isolation, molecular identification, and probiotic characterization of Lactobacillus and Lactococcus strains from traditional Iranian dairy products. Primary probiotic assessments indicated high tolerance to low pH and high bile salt conditions, high anti-pathogenic activities, and susceptibility to high consumption antibiotics, thus proving that both strains possess probiotic potential. Cytotoxicity assessments were used to analyze the effects of the secreted metabolite on different cancer cell lines, including HT29, AGS, MCF-7, and HeLa, as well as a normal human cell line (HUVEC). Results showed acceptable cytotoxic properties for secreted metabolites (40 μg/ml dry weight) of Lactococcus lactis subsp. Lactis 44Lac. Such performance was similar to that of Taxol against all of the treated cancer cell lines; however, the strain exhibited no toxicity on the normal cell line. Cytotoxic assessments through flow cytometry and fluorescent microscopy demonstrated that apoptosis is the main cytotoxic mechanism for secreted metabolites of L. lactis subsp. Lactis 44Lac. By contrast, the effects of protease-treated metabolites on the AGS cell line verified the protein nature of anti-cancer metabolites. However, precise characterizations and in vitro/in vivo investigations on purified proteins should be conducted before these metabolites are introduced as potential anti-cancer therapeutics. PMID:25168457

  4. L’infection bactérienne chez le patient brûlé

    PubMed Central

    Le Floch, R.; Naux, E.; Arnould, J.F.

    2015-01-01

    Summary La mort d’un patient brûlé est le plus souvent causée par une infection, bactérienne dans la grande majorité des cas. La perte de la barrière cutanée, les dispositifs invasifs et l’immunodépression liée à la brûlure sont trois mécanismes concourant à la survenue de ces infections. Chez un patient inflammatoire, les signes infectieux généraux d’infection sont peu discriminants. Du fait de la gravité des infections chez ce patient, leur prévention est un paramètre essentiel de la prise en charge. En raison des particularités pharmacocinétiques des brûlés, les posologies d’antibiotiques doivent être adaptés et les dosages sanguins doivent être systématiques. A l’heure où les résistances deviennent préoccupantes, les recherches sur les thérapeutiques sur les alternatives thérapeutiques parmi lesquels les inhibiteurs de facteurs de virulence, les peptides antimicrobiens, les polyphénols, l’immunothérapie…) deviennent cruciales. L’une des possibilités thérapeutiques les plus prometteuses semble être la phagothérapie. PMID:27252607

  5. Changes in ciprofloxacin resistance levels in Enterobacter aerogenes isolates associated with variable expression of the aac(6')-Ib-cr gene.

    PubMed

    Ruiz, Elena; Ocampo-Sosa, Alain A; Alcoba-Flórez, Julia; Román, Elena; Arlet, Guillaume; Torres, Carmen; Martínez-Martínez, Luis

    2012-02-01

    Two closely related Enterobacter aerogenes isolates presented a new identical aac(6')-Ib-cr genetic environment, including IS26. One isolate showed lower MICs of ciprofloxacin, norfloxacin, tobramycin, and amikacin and decreased expression of aac(6')-Ib-cr, which might be related to a 12-bp deletion causing a displacement of the -10 box upstream of the aac(6')-Ib-cr gene.

  6. Draft Genome Assemblies of Enterobacter aerogenes CDC 6003-71, Enterobacter cloacae CDC 442-68, and Pantoea agglomerans UA 0804-01.

    PubMed

    Minogue, T D; Daligault, H E; Davenport, K W; Bishop-Lilly, K A; Bruce, D C; Chain, P S; Coyne, S R; Chertkov, O; Freitas, T; Frey, K G; Jaissle, J; Koroleva, G I; Ladner, J T; Palacios, G F; Redden, C L; Xu, Y; Johnson, S L

    2014-10-23

    The Enterobacteriaceae are environmental and enteric microbes. We sequenced the genomes of two Enterobacter reference strains, E. aerogenes CDC 6003-71 and E. cloacae CDC 442-68, as well as one near neighbor used as an exclusionary reference for diagnostics, Pantoea agglomerans CDC UA0804-01. The genome sizes range from 4.72 to 5.55 Mbp and have G+C contents from 54.6 to 55.1%.

  7. Profil bactériologique du pied diabétique et son impact sur le choix des antibiotiques

    PubMed Central

    Zemmouri, Adil; Tarchouli, Mohamed; Benbouha, Abdellatif; Lamkinsi, Tarik; Bensghir, Mustapha; Elouennass, Mostafa; Haimeur, Cherqui

    2015-01-01

    Introduction Analyse du profil bactériologique des pieds diabétiques pris en charge à l'hôpital militaire de Rabat et son influence sur l'antibiothérapie de première intention. Méthodes Etude prospective non randomisée étalée sur 18 mois, ayant concerné 105 patients. Après recueil des données et en attente des résultats bactériologiques nos patients ont été divisés en deux groupes: un groupe a été mis sous Amoxicilline + Acide clavulanique + Gentamycine (59 patients) et un groupe sous Ertapénème±Gentamycine (46 patients). Résultats L’étude a regroupé 85 hommes et 20 femmes (sexe ratio = 4.26). L’âge moyen est de 64.4 ans. La gangrène a été observée chez 79% des malades; elle était humide-donc surinfectée en principe- dans 43% des cas. Par ailleurs, 67% des malades ont un chiffre de globules blancs 12000 définissant une infection sévère. L'ostéolyse a été mise en évidence chez 27% de nos patients. Parmi les différentes techniques de prélèvements: 81% ont été profonds dont 21% de biopsie osseuse per opératoire et 14% de prélèvements combinés. 42% de ces prélèvements sont poly microbiens et 21% sont stériles. Les résultats bactériologiques viennent confirmer la prédominance des bactéries aérobies à Gram positif. Le taux de remplacement de l'Ertapénème est de 22% contre un taux de 50% pour l'Amoxiclav. Conclusion L'antibiothérapie ne doit être instaurée qu'en cas d'infection du pied diabétique diagnostiquée sur les critères cliniques établis par les consensus internationaux récents. Le respect des mesures de lutte contre la diffusion de la résistance bactérienne s'avère primordiale. PMID:27386024

  8. Integrated evaluation of aerogenic pollution by air-transported heavy metals (Pb, Cd, Ni, Zn, Mn and Cu) in the analysis of the main deposit media.

    PubMed

    Baltrėnaitė, Edita; Baltrėnas, Pranas; Lietuvninkas, Arvydas; Serevičienė, Vaida; Zuokaitė, Eglė

    2014-01-01

    The composition of the ambient air is constantly changing; therefore, the monitoring of ambient air quality to detect the changes caused by aerogenic pollutants makes the essential part of general environmental monitoring. To achieve more effective improvement of the ambient air quality, the Directive 2008/50/EC on 'Ambient Air Quality and Cleaner Air for Europe' was adopted by the European Parliament and the European Council. It informed the public and enterprises about a negative effect of pollution on humans, animals and plants, as well as about the need for monitoring aerogenic pollutants not only at the continuous monitoring stations but also by using indicator methods, i.e. by analysing natural deposit media. The problem of determining the relationship between the accumulation level of pollutants by a deposit medium and the level of air pollution and its risks is constantly growing in importance. The paper presents a comprehensive analysis of the response of the main four deposit media, i.e. snow cover, soil, pine bark and epigeic mosses, to the long-term pollution by aerogenic pollutants which can be observed in the area of oil refinery influence. Based on the quantitative expressions of the amounts of the accumulated pollutants in the deposit media, the territory of the oil refinery investigated in this paper has been referred to the areas of mild or moderate pollution.

  9. Transcriptome Analysis Reveals Mechanisms by Which Lactococcus lactis Acquires Nisin Resistance

    PubMed Central

    Kramer, Naomi E.; van Hijum, Sacha A. F. T.; Knol, Jan; Kok, Jan; Kuipers, Oscar P.

    2006-01-01

    Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nisr, which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane. PMID:16641446

  10. Deciphering a unique biotin scavenging pathway with redundant genes in the probiotic bacterium Lactococcus lactis

    PubMed Central

    Zhang, Huimin; Wang, Qingjing; Fisher, Derek J.; Cai, Mingzhu; Chakravartty, Vandana; Ye, Huiyan; Li, Ping; Solbiati, Jose O.; Feng, Youjun

    2016-01-01

    Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment. The in vivo function of the two L. lactis birA genes was judged by their abilities to complement the conditional lethal E. coli birA mutant. Thin-layer chromatography and mass spectroscopy assays demonstrated that these two recombinant BirA proteins catalyze the biotinylation reaction of the acceptor biotin carboxyl carrier protein (BCCP), through the expected biotinoyl-AMP intermediate. Gel shift assays were used to characterize bioY1_LL and BirA1_LL. We also determined the ability to uptake 3H-biotin by L. lactis. Taken together, our results deciphered a unique biotin scavenging pathway with redundant genes present in the probiotic bacterium L. lactis. PMID:27161258

  11. The Prophylactic Effect of Probiotic Enterococcus lactis IW5 against Different Human Cancer Cells

    PubMed Central

    Nami, Yousef; Haghshenas, Babak; Haghshenas, Minoo; Abdullah, Norhafizah; Yari Khosroushahi, Ahmad

    2015-01-01

    Enterococcus lactis IW5 was obtained from human gut and the potential probiotic characteristics of this organism were then evaluated. Results showed that this strain was highly resistant to low pH and high bile salt and adhered strongly to Caco-2 human epithelial colorectal cell lines. The supernatant of E. lactis IW5 strongly inhibited the growth of several pathogenic bacteria and decreased the viability of different cancer cells, such as HeLa, MCF-7, AGS, HT-29, and Caco-2. Conversely, E. lactis IW5 did not inhibit the viability of normal FHs-74 cells. This strain did not generate toxic enzymes, including β-glucosidase, β-glucuronidase, and N-acetyl-β-glucosaminidase and was highly susceptible to ampicillin, gentamycin, penicillin, vancomycin, clindamycin, sulfamethoxazol, and chloramphenicol but resistant to erythromycin and tetracyclin. This study provided evidence for the effect of E. lactis IW5 on cancer cells. Therefore, E. lactis IW5, as a bioactive therapeutics, should be subjected to other relevant tests to verify the therapeutic suitability of this strain for clinical applications. PMID:26635778

  12. Induction of Sucrose Utilization Genes from Bifidobacterium lactis by Sucrose and Raffinose

    PubMed Central

    Trindade, Marla I.; Abratt, Valerie R.; Reid, Sharon J.

    2003-01-01

    The probiotic organism Bifidobacterium lactis was isolated from a yoghurt starter culture with the aim of analyzing its use of carbohydrates for the development of prebiotics. A sucrose utilization gene cluster of B. lactis was identified by complementation of a gene library in Escherichia coli. Three genes, encoding a sucrose phosphorylase (ScrP), a GalR-LacI-type transcriptional regulator (ScrR), and a sucrose transporter (ScrT), were identified by sequence analysis. The scrP gene was expressed constitutively from its own promoter in E. coli grown in complete medium, and the strain hydrolyzed sucrose in a reaction that was dependent on the presence of phosphates. Primer extension experiments with scrP performed by using RNA isolated from B. lactis identified the transcriptional start site 102 bp upstream of the ATG start codon, immediately adjacent to a palindromic sequence resembling a regulator binding site. In B. lactis, total sucrase activity was induced by the presence of sucrose, raffinose, or oligofructose in the culture medium and was repressed by glucose. RNA analysis of the scrP, scrR, and scrT genes in B. lactis indicated that expression of these genes was influenced by transcriptional regulation and that all three genes were similarly induced by sucrose and raffinose and repressed by glucose. Analysis of the sucrase activities of deletion constructs in heterologous E. coli indicated that ScrR functions as a positive regulator. PMID:12513973

  13. Enterococcus lactis sp. nov., from Italian raw milk cheeses.

    PubMed

    Morandi, Stefano; Cremonesi, Paola; Povolo, Milena; Brasca, Milena

    2012-08-01

    Ten atypical Enterococcus strains were isolated from Italian raw milk cheeses. The 16S rRNA gene, phenylalanyl-tRNA synthase alpha subunit (pheS), RNA polymerase alpha subunit (rpoA) and the 16S-23S rRNA intergenic transcribed spacer (ITS) sequences, randomly amplified polymorphic DNA (RAPD) PCR and the phenotypic properties revealed that the isolates represent a novel enterococcal species. On the basis of 16S rRNA gene sequence analysis, the isolates were closely related to Enterococcus hirae ATCC 8043(T), Enterococcus durans CECT 411(T) and Enterococcus faecium ATCC 19434(T), with 98.8, 98.9 and 99.4% sequence similarity, respectively. On the basis of sequence analysis of the housekeeping gene pheS, the reference strain, BT159(T), occupied a position separate from E. faecium LMG 16198. The group of isolates could be easily differentiated from recognized species of the genus Enterococcus by 16S-23S rRNA ITS analysis, RAPD-PCR and phenotypic characteristics. The name Enterococcus lactis sp. nov. is proposed, with BT159(T) ( = DSM 23655(T) = LMG 25958(T)) as the type strain.

  14. Dynamic modeling of lactic acid fermentation metabolism with Lactococcus lactis.

    PubMed

    Oh, Euhlim; Lu, Mingshou; Park, Changhun; Park, Changhun; Oh, Han Bin; Lee, Sang Yup; Lee, Jinwon

    2011-02-01

    A dynamic model of lactic acid fermentation using Lactococcus lactis was constructed, and a metabolic flux analysis (MFA) and metabolic control analysis (MCA) were performed to reveal an intensive metabolic understanding of lactic acid bacteria (LAB). The parameter estimation was conducted with COPASI software to construct a more accurate metabolic model. The experimental data used in the parameter estimation were obtained from an LC-MS/ MS analysis and time-course simulation study. The MFA results were a reasonable explanation of the experimental data. Through the parameter estimation, the metabolic system of lactic acid bacteria can be thoroughly understood through comparisons with the original parameters. The coefficients derived from the MCA indicated that the reaction rate of L-lactate dehydrogenase was activated by fructose 1,6-bisphosphate and pyruvate, and pyruvate appeared to be a stronger activator of L-lactate dehydrogenase than fructose 1,6-bisphosphate. Additionally, pyruvate acted as an inhibitor to pyruvate kinase and the phosphotransferase system. Glucose 6-phosphate and phosphoenolpyruvate showed activation effects on pyruvate kinase. Hexose transporter was the strongest effector on the flux through L-lactate dehydrogenase. The concentration control coefficient (CCC) showed similar results to the flux control coefficient (FCC).

  15. Lignes directrices canadiennes sur la rhinosinusite bactérienne aiguë

    PubMed Central

    Kaplan, Alan

    2014-01-01

    Résumé Objectif Faire un résumé clinique des lignes directrices canadiennes sur la rhinosinusite bactérienne aiguë (RSBA) qui présente des éléments d’intérêt pour les médecins de famille. Source des données Les auteurs des lignes directrices ont effectué une recherche documentaire systématique et ont rédigé des recommandations. Une cote a été donnée à la fois en fonction de la fiabilité des données probantes et de la solidité des recommandations. On a sollicité les commentaires d’experts en la matière venant de l’extérieur, ainsi que l’aval de sociétés médicales canadiennes (Association pour la microbiologie médicale et l’infectiologie Canada, Société canadienne d’allergie et d’immunologie clinique, Société canadienne d’otorhinolaryngologie et de chirurgie cervicofaciale, Association canadienne des médecins d’urgence et Regroupement canadien des médecins de famille en santé respiratoire). Message principal Le diagnostic de la RSBA repose sur la présence de symptômes particuliers et leur durée; l’imagerie ou une culture n’est pas nécessaire dans les cas peu compliqués. Le traitement dépend de la gravité des symptômes, notamment avec des corticostéroïdes intranasaux (CSIN) recommandés comme monothérapie pour les cas de légers à modérés, quoique leurs bienfaits soient modestes. Le recours à des CSIN accompagnés d’antibiotiques est réservé aux patients qui ne répondent pas aux CSIN après 72 heures et comme traitement initial des patients dont les symptômes sont graves. Le choix de l’antibiotique doit tenir compte du pathogène soupçonné, du risque de résistance, des problèmes concomitants et des tendances locales de la résistance aux antimicrobiens. Des thérapies d’appoint comme l’irrigation nasale avec une solution saline sont recommandées. En présence de cas réfractaires au traitement, d’épisodes récurrents et de signes de complications, on devrait demander une

  16. Unity in organisation and regulation of catabolic operons in Lactobacillus plantarum, Lactococcus lactis and Listeria monocytogenes.

    PubMed

    Andersson, Ulrika; Molenaar, Douwe; Rådström, Peter; de Vos, Willem M

    2005-04-01

    Global regulatory circuits together with more specific local regulators play a notable role when cells are adapting to environmental changes. Lactococcus lactis is a lactic acid bacterium abundant in nature fermenting most mono- and disaccharides. Comparative genomics analysis of the operons encoding the proteins and enzymes crucial for catabolism of lactose, maltose and threhalose revealed an obvious unity in operon organisation . The local regulator of each operon was located in a divergent transcriptional direction to the rest of the operon including the transport protein-encoding genes. Furthermore, in all three operons a catabolite responsive element (CRE) site was detected inbetween the gene encoding the local regulator and one of the genes encoding a sugar transport protein. It is evident that regardless of type of transport system and catabolic enzymes acting upon lactose, maltose and trehalose, respectively, Lc. lactis shows unity in both operon organisation and regulation of these catabolic operons. This knowledge was further extended to other catabolic operons in Lc. lactis and the two related bacteria Lactobacillus plantarum and Listeria monocytogenes. Thirty-nine catabolic operons responsible for degradation of sugars and sugar alcohols in Lc. lactis, Lb. plantarum and L. monocytogenes were investigated and the majority of those possessed the same organisation as the lactose, maltose and trehalose operons of Lc. lactis. Though, the frequency of CRE sites and their location varied among the bacteria. Both Lc. lactis and Lb. plantarum showed CRE sites in direct proximity to genes coding for proteins responsible for sugar uptake. However, in L. monocytogenes CRE sites were not frequently found and not in the vicinity of genes encoding transport proteins, suggesting a more local mode of regulation of the catabolic operons found and/or the use of inducer control in this bacterium. PMID:15900965

  17. Dechlorination of 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane by Aerobacter aerogenes

    USGS Publications Warehouse

    Wedemeyer, Gary

    1967-01-01

    Whole cells or cell-free extracts of Aerobacter aerogenes catalyze the degradation of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in vitro to at least seven metabolites: 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE); 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD); 1-chloro-2,2-bis(p-chlorophenyl)ethylene (DDMU); 1-chloro-2,2-bis(p-chlorophenyl)ethane (DDMS); unsym-bis(p-chlorophenyl)ethylene (DDNU); 2,2-bis(p-chlorophenyl)acetate (DDA); and 4,4′-dichlorobenzophenone (DBP). The use of metabolic inhibitors together with pH and temperature studies indicated that discrete enzymes are involved. By use of the technique of sequential analysis, the metabolic pathway was shown to be: DDT → DDD →DDMU →DDMS → DDNU → DDA → DBP, or DDT → DDE. Dechlorination was marginally enhanced by light-activated flavin mononucleotide.

  18. Phenolic compounds: Strong inhibitors derived from lignocellulosic hydrolysate for 2,3-butanediol production by Enterobacter aerogenes.

    PubMed

    Lee, Sang Jun; Lee, Ju Hun; Yang, Xiaoguang; Kim, Sung Bong; Lee, Ja Hyun; Yoo, Hah Young; Park, Chulhwan; Kim, Seung Wook

    2015-12-01

    Lignocellulosic biomass are attractive feedstocks for 2,3-butanediol production due to their abundant supply and low price. During the hydrolysis of lignocellulosic biomass, various byproducts are formed and their effects on 2,3-butanediol production were not sufficiently studied compared to ethanol production. Therefore, the effects of compounds derived from lignocellulosic biomass (weak acids, furan derivatives and phenolics) on the cell growth, the 2,3-butanediol production and the enzymes activity involved in 2,3-butanediol production were evaluated using Enterobacter aerogenes ATCC 29007. The phenolic compounds showed the most toxic effects on cell growth, 2,3-butanediol production and enzyme activity, followed by furan derivatives and weak acids. The significant effects were not observed in the presence of acetic acid and formic acid. Also, feasibility of 2,3-butanediol production from lignocellulosic biomass was evaluated using Miscanthus as a feedstock. In the fermentation of Miscanthus hydrolysate, 11.00 g/L of 2,3-butanediol was obtained from 34.62 g/L of reducing sugar. However, 2,3-butanediol was not produced when the concentration of total phenolic compounds in the hydrolysate increased to more than 1.5 g/L. The present study provides useful information to develop strategies for biological production of 2,3-butanediol and to establish biorefinery for biochemicals from lignocellulosic biomass. PMID:26479290

  19. Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD+-dependent formate dehydrogenase.

    PubMed

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Wu, Xi; Xing, Xin-Hui

    2010-03-01

    The NAD+-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.

  20. Mutational analysis of the hyc-operon determining the relationship between hydrogenase-3 and NADH pathway in Enterobacter aerogenes.

    PubMed

    Pi, Jian; Jawed, Muhammad; Wang, Jun; Xu, Li; Yan, Yunjun

    2016-01-01

    In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3.

  1. Disruption of lactate dehydrogenase and alcohol dehydrogenase for increased hydrogen production and its effect on metabolic flux in Enterobacter aerogenes.

    PubMed

    Zhao, Hongxin; Lu, Yuan; Wang, Liyan; Zhang, Chong; Yang, Cheng; Xing, Xinhui

    2015-10-01

    Hydrogen production by Enterobacter aerogenes from glucose was enhanced by deleting the targeted ldhA and adh genes responsible for two NADH-consuming pathways which consume most NADH generated from glycolysis. Compared with the wild-type, the hydrogen yield of IAM1183-ΔldhA increased 1.5 fold. Metabolic flux analysis showed both IAM1183-ΔldhA and IAM1183-Δadh exhibited significant changes in flux, including enhanced flux towards the hydrogen generation. The lactate production of IAM1183-ΔldhA significantly decreased by 91.42%, while the alcohol yield of IAM1183-Δadh decreased to 30%. The mutant IAM1183-ΔldhA with better hydrogen-producing performance was selected for further investigation in a 5-L fermentor. The hydrogen production of IAM1183-ΔldhA was 2.3 times higher than the wild-type. Further results from the fermentation process showed that the pH decreased to 5.39 levels, then gradually increased to 5.96, indicating that some acidic metabolites might be degraded or uptaken by cells.

  2. Phenolic compounds: Strong inhibitors derived from lignocellulosic hydrolysate for 2,3-butanediol production by Enterobacter aerogenes.

    PubMed

    Lee, Sang Jun; Lee, Ju Hun; Yang, Xiaoguang; Kim, Sung Bong; Lee, Ja Hyun; Yoo, Hah Young; Park, Chulhwan; Kim, Seung Wook

    2015-12-01

    Lignocellulosic biomass are attractive feedstocks for 2,3-butanediol production due to their abundant supply and low price. During the hydrolysis of lignocellulosic biomass, various byproducts are formed and their effects on 2,3-butanediol production were not sufficiently studied compared to ethanol production. Therefore, the effects of compounds derived from lignocellulosic biomass (weak acids, furan derivatives and phenolics) on the cell growth, the 2,3-butanediol production and the enzymes activity involved in 2,3-butanediol production were evaluated using Enterobacter aerogenes ATCC 29007. The phenolic compounds showed the most toxic effects on cell growth, 2,3-butanediol production and enzyme activity, followed by furan derivatives and weak acids. The significant effects were not observed in the presence of acetic acid and formic acid. Also, feasibility of 2,3-butanediol production from lignocellulosic biomass was evaluated using Miscanthus as a feedstock. In the fermentation of Miscanthus hydrolysate, 11.00 g/L of 2,3-butanediol was obtained from 34.62 g/L of reducing sugar. However, 2,3-butanediol was not produced when the concentration of total phenolic compounds in the hydrolysate increased to more than 1.5 g/L. The present study provides useful information to develop strategies for biological production of 2,3-butanediol and to establish biorefinery for biochemicals from lignocellulosic biomass.

  3. Host-pathogen interactions in specific pathogen-free chickens following aerogenous infection with Chlamydia psittaci and Chlamydia abortus.

    PubMed

    Kalmar, Isabelle; Berndt, Angela; Yin, Lizi; Chiers, Koen; Sachse, Konrad; Vanrompay, Daisy

    2015-03-15

    Although Chlamydia (C.) psittaci infections are recognized as an important factor causing economic losses and impairing animal welfare in poultry production, the specific mechanisms leading to severe clinical outcomes are poorly understood. In the present study, we comparatively investigated pathology and host immune response, as well as systemic dissemination and expression of essential chlamydial genes in the course of experimental aerogeneous infection with C. psittaci and the closely related C. abortus, respectively, in specific pathogen-free chicks. Clinical signs appeared sooner and were more severe in the C. psittaci-infected group. Compared to C. abortus infection, more intense systemic dissemination of C. psittaci correlated with higher and faster infiltration of immune cells, as well as more macroscopic lesions and epithelial pathology, such as hyperplasia and erosion. In thoracic air sac tissue, mRNA expression of immunologically relevant factors, such as IFN-γ, IL-1β, IL-6, IL-17, IL-22, LITAF and iNOS was significantly stronger up-regulated in C. psittaci- than in C. abortus-infected birds between 3 and 14 days post-infection. Likewise, transcription rates of the chlamydial genes groEL, cpaf and ftsW were consistently higher in C. psittaci during the acute phase. These findings illustrate that the stronger replication of C. psittaci in its natural host also evoked a more intense immune response than in the case of C. abortus infection.

  4. Yeast on the milky way: genetics, physiology and biotechnology of Kluyveromyces lactis.

    PubMed

    Rodicio, Rosaura; Heinisch, Jürgen J

    2013-05-01

    The milk yeast Kluyveromyces lactis has a life cycle similar to that of Saccharomyces cerevisiae and can be employed as a model eukaryote using classical genetics, such as the combination of desired traits, by crossing and tetrad analysis. Likewise, a growing set of vectors, marker cassettes and tags for fluorescence microscopy are available for manipulation by genetic engineering and investigating its basic cell biology. We here summarize these applications, as well as the current knowledge regarding its central metabolism, glucose and extracellular stress signalling pathways. A short overview on the biotechnological potential of K. lactis concludes this review.

  5. Mycotoxin production by isolates of Fusarium lactis from greenhouse sweet pepper (Capsicum annuum).

    PubMed

    Yang, Y; Bouras, N; Yang, J; Howard, R J; Strelkov, S E

    2011-12-01

    Internal fruit rot, caused by Fusarium lactis, is an important disease of sweet pepper (Capsicum annuum) in Canadian greenhouses. Production of the mycotoxins fumonisin B₁ (FB₁), moniliformin (MON) and beauvericin (BEA) by F. lactis (17 isolates) and the related species F. proliferatum (three isolates) and F. verticillioides (one isolate), which are also associated with internal fruit rot, was evaluated on rice medium. All 21 isolates examined were found to produce BEA, at concentrations ranging from 13.28 to 1674.60 ppm, while 13 of 17 F. lactis isolates and two of three F. proliferatum isolates produced MON (0.23 to 181.85 ppm). Only one isolate of F. lactis produced detectable levels of FB₁ in culture, whereas all three F. proliferatum isolates and the F. verticilloides isolate produced this mycotoxin (0.28 to 314 ppm). Production of FB₁, MON and BEA was also evaluated in inoculated pepper fruits showing mild or severe symptoms of infection. FB₁ could be detected in both lightly and heavily diseased fruit tissue after inoculation with F. lactis, F. proliferatum or F. verticilloides, at concentrations ranging from 0.61 to 8.04 ppm. BEA was also detected in lightly and heavily diseased fruit tissue inoculated with F. lactis, as well as in heavily diseased tissue inoculated with F. proliferatum (3.00 to 19.43 ppm), but not in tissue inoculated with F. verticilloides. MON was detected in all tissues inoculated with F. proliferatum or F. verticilloides, and in heavily diseased tissue inoculated with F. lactis (0.03 to 0.27 ppm). The three mycotoxins were also found in naturally infected sweet pepper fruits exhibiting symptoms of internal fruit rot and collected from a commercial greenhouse. The production of MON, BEA and FB₁ alone or in combination by isolates of F. lactis suggests that development of internal fruit rot of sweet pepper is an important food safety concern, and that every effort should be made to cull infected fruit before it makes it to

  6. Yeast on the milky way: genetics, physiology and biotechnology of Kluyveromyces lactis.

    PubMed

    Rodicio, Rosaura; Heinisch, Jürgen J

    2013-05-01

    The milk yeast Kluyveromyces lactis has a life cycle similar to that of Saccharomyces cerevisiae and can be employed as a model eukaryote using classical genetics, such as the combination of desired traits, by crossing and tetrad analysis. Likewise, a growing set of vectors, marker cassettes and tags for fluorescence microscopy are available for manipulation by genetic engineering and investigating its basic cell biology. We here summarize these applications, as well as the current knowledge regarding its central metabolism, glucose and extracellular stress signalling pathways. A short overview on the biotechnological potential of K. lactis concludes this review. PMID:23576126

  7. Short- and long-term dynamics in the intestinal microbiota following ingestion of Bifidobacterium animalis subsp. lactis GCL2505

    PubMed Central

    TANAKA, Yoshiyuki; TAKAMI, Kazuyo; NISHIJIMA, Tomohiko; AOKI, Ryo; MAWATARI, Takashi; IKEDA, Takayuki

    2015-01-01

    Bifidobacterium animalis subsp. lactis GCL2505 (B. lactis GCL2505) is able to survive passage through the intestines and proliferate. The daily dynamics of the intestinal bifidobacteria following ingestion of probiotics are not yet clear. Moreover, the effects of long-term ingestion of probiotics on the intestinal microbiota have not been well studied. Two experiments were performed in the present study. In Experiment 1, 53 healthy female volunteers received B. lactis GCL2505; B. bifidum GCL2080, which can survive but not proliferate in the intestine; or yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus for 2 weeks, and the daily dynamics of intestinal bifidobacteria were investigated. The number of fecal bifidobacteria significantly increased on day 1, and this was maintained until day 14 in the B. lactis GCL2505 ingestion group. However, no significant change in the number of fecal bifidobacteria was observed in the other groups throughout the ingestion period. In Experiment 2, 38 constipated volunteers received either B. lactis GCL2505 or a placebo for 8 weeks. Both the number of fecal bifidobacteria and the frequency of defecation significantly increased throughout the ingestion period in the B. lactis GCL2505 ingestion group. These results suggested that the proliferation of ingested bifidobacteria within the intestine contributed to a rapid increase in the amount of intestinal bifidobacteria and subsequent maintenance of these levels. Moreover, B. lactis GCL2505 improved the intestinal microbiota more effectively than non-proliferating bifidobacteria and lactic acid bacteria. PMID:26594607

  8. Short- and long-term dynamics in the intestinal microbiota following ingestion of Bifidobacterium animalis subsp. lactis GCL2505.

    PubMed

    Tanaka, Yoshiyuki; Takami, Kazuyo; Nishijima, Tomohiko; Aoki, Ryo; Mawatari, Takashi; Ikeda, Takayuki

    2015-01-01

    Bifidobacterium animalis subsp. lactis GCL2505 (B. lactis GCL2505) is able to survive passage through the intestines and proliferate. The daily dynamics of the intestinal bifidobacteria following ingestion of probiotics are not yet clear. Moreover, the effects of long-term ingestion of probiotics on the intestinal microbiota have not been well studied. Two experiments were performed in the present study. In Experiment 1, 53 healthy female volunteers received B. lactis GCL2505; B. bifidum GCL2080, which can survive but not proliferate in the intestine; or yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus for 2 weeks, and the daily dynamics of intestinal bifidobacteria were investigated. The number of fecal bifidobacteria significantly increased on day 1, and this was maintained until day 14 in the B. lactis GCL2505 ingestion group. However, no significant change in the number of fecal bifidobacteria was observed in the other groups throughout the ingestion period. In Experiment 2, 38 constipated volunteers received either B. lactis GCL2505 or a placebo for 8 weeks. Both the number of fecal bifidobacteria and the frequency of defecation significantly increased throughout the ingestion period in the B. lactis GCL2505 ingestion group. These results suggested that the proliferation of ingested bifidobacteria within the intestine contributed to a rapid increase in the amount of intestinal bifidobacteria and subsequent maintenance of these levels. Moreover, B. lactis GCL2505 improved the intestinal microbiota more effectively than non-proliferating bifidobacteria and lactic acid bacteria. PMID:26594607

  9. Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ †

    PubMed Central

    Ladero, Victor; Rattray, Fergal P.; Mayo, Baltasar; Martín, María Cruz; Fernández, María; Alvarez, Miguel A.

    2011-01-01

    Lactococcus lactis is a prokaryotic microorganism with great importance as a culture starter and has become the model species among the lactic acid bacteria. The long and safe history of use of L. lactis in dairy fermentations has resulted in the classification of this species as GRAS (General Regarded As Safe) or QPS (Qualified Presumption of Safety). However, our group has identified several strains of L. lactis subsp. lactis and L. lactis subsp. cremoris that are able to produce putrescine from agmatine via the agmatine deiminase (AGDI) pathway. Putrescine is a biogenic amine that confers undesirable flavor characteristics and may even have toxic effects. The AGDI cluster of L. lactis is composed of a putative regulatory gene, aguR, followed by the genes (aguB, aguD, aguA, and aguC) encoding the catabolic enzymes. These genes are transcribed as an operon that is induced in the presence of agmatine. In some strains, an insertion (IS) element interrupts the transcription of the cluster, which results in a non-putrescine-producing phenotype. Based on this knowledge, a PCR-based test was developed in order to differentiate nonproducing L. lactis strains from those with a functional AGDI cluster. The analysis of the AGDI cluster and their flanking regions revealed that the capacity to produce putrescine via the AGDI pathway could be a specific characteristic that was lost during the adaptation to the milk environment by a process of reductive genome evolution. PMID:21803900

  10. Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis.

    PubMed

    Yagnik, Bhrugu; Padh, Harish; Desai, Priti

    2016-04-01

    Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells. PMID:26655884

  11. Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis.

    PubMed

    Yagnik, Bhrugu; Padh, Harish; Desai, Priti

    2016-04-01

    Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells.

  12. Use of a Genetically Enhanced, Pediocin-Producing Starter Culture, Lactococcus lactis subsp. lactis MM217, To Control Listeria monocytogenes in Cheddar Cheese

    PubMed Central

    Buyong, Nurliza; Kok, Jan; Luchansky, John B.

    1998-01-01

    Cheddar cheese was prepared with Lactococcus lactis subsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 106 CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 103 CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8°C for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 107 CFU per g after 2 weeks of ripening and then gradually decreased to about 103 CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 102 CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes. PMID:9835572

  13. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

    PubMed Central

    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2015-01-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

  14. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2015-03-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

  15. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2015-03-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

  16. Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis

    PubMed Central

    González-Siso, M Isabel; García-Leiro, Ana; Tarrío, Nuria; Cerdán, M Esperanza

    2009-01-01

    A lot of studies have been carried out on Saccharomyces cerevisiae, an yeast with a predominant fermentative metabolism under aerobic conditions, which allows exploring the complex response induced by oxidative stress. S. cerevisiae is considered a eukaryote model for these studies. We propose Kluyveromyces lactis as a good alternative model to analyse variants in the oxidative stress response, since the respiratory metabolism in this yeast is predominant under aerobic conditions and it shows other important differences with S. cerevisiae in catabolic repression and carbohydrate utilization. The knowledge of oxidative stress response in K. lactis is still a developing field. In this article, we summarize the state of the art derived from experimental approaches and we provide a global vision on the characteristics of the putative K. lactis components of the oxidative stress response pathway, inferred from their sequence homology with the S. cerevisiae counterparts. Since K. lactis is also a well-established alternative host for industrial production of native enzymes and heterologous proteins, relevant differences in the oxidative stress response pathway and their potential in biotechnological uses of this yeast are also reviewed. PMID:19715615

  17. Isolation of a recombination-deficient mutant of Streptococcus lactis ML3.

    PubMed Central

    Anderson, D G; McKay, L L

    1983-01-01

    A recombination-deficient mutant of Streptococcus lactis ML3 designated MMS36 was isolated on the basis of its sensitivity to methyl methanesulfonate. This mutant also displayed sensitivity to UV irradiation. The inability of MMS36 to mediate homologous recombination was demonstrated by transduction of plasmid-linked lactose fermenting ability but not chromosomally mediated streptomycin resistance. PMID:6409888

  18. Oral immunization of mice with Lactococcus lactis expressing the rotavirus VP8* protein.

    PubMed

    Rodríguez-Díaz, Jesús; Montava, Rebeca; Viana, Rosa; Buesa, Javier; Pérez-Martínez, Gaspar; Monedero, Vicente

    2011-06-01

    The efficacy of recombinant Lactococcus lactis as a delivery vehicle for a rotavirus antigen was evaluated in a mouse model. The rotavirus VP8* protein was expressed intracellularly and extracellularly in L. lactis wild type and in an alr mutant deficient in alanine racemase activity, necessary for the synthesis of the cell-wall component D: -alanine. When the mucosal immune response was evaluated by measuring VP8*-specific IgA antibody in faeces, wild-type L. lactis triggered a low IgA synthesis only when the secreting strain was used. In contrast, VP8*-specific IgA was detected in faeces of both groups of mice orally given the alr mutant expressing extracellular VP8* and intracellular VP8*, which reached levels similar to that obtained with the wild type secreting strain. However, oral administration of the recombinant strains did not induce serum IgG or IgA responses. L. lactis cell-wall mutants may therefore provide certain advantages when low-antigenic proteins are expressed intracellularly. However, the low immune response obtained by using this antigen-bacterial host combination prompts to the use of new strains and vaccination protocols in order to develop acceptable rotavirus immunization levels.

  19. 21 CFR 184.1388 - Lactase enzyme preparation from Kluyveromyces lactis.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) This enzyme preparation is derived from the nonpathogenic, nontoxicogenic yeast Kluyveromyces lactis... 683), which converts lactose to glucose and galactose. It is prepared from yeast that has been grown in a pure culture fermentation and by using materials that are generally recognized as safe or...

  20. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose in Lactococcus lactis using a nisin-controlled gene expression system. Production of DsrI was optimized using several different background vectors, signal peptides, str...

  1. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  2. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  3. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  4. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave. NW... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Aminopeptidase enzyme preparation derived from... Aminopeptidase enzyme preparation derived from lactococcus lactis. (a) Aminopeptidase enzyme preparation...

  5. Dealing with different methods for Kluyveromyces lactis β-galactosidase purification

    PubMed Central

    Becerra, M; Cerdãn, E

    1998-01-01

    Several micro-scale chromatography-based procedures for purification of the β-galactosidase from the yeast Kluyveromyces lactis were assayed. Purified enzyme was suitable to be used as antigen to induce polyclonal antibodies production. Specific staining of non-denaturing PAGE gels with chromogenic substrates allowed the determination of the number of subunits forming the native enzyme. PMID:12734592

  6. A Case of Infective Endocarditis and Pulmonary Septic Emboli Caused by Lactococcus lactis

    PubMed Central

    Habib, Adib; Asli, Nazih; Geffen, Yuval; Miron, Dan; Elias, Nael

    2016-01-01

    Infective endocarditis is a rare condition in children with normal hearts. We present here a case of previously healthy eleven-year-old girl with infective endocarditis and pulmonary septic emboli caused by a very rare bacterial etiology (Lactococcus lactis). Identification of this pathogen was only made by polymerase chain reaction. PMID:27774332

  7. Genome Sequence of Lactococcus lactis subsp. cremoris Mast36, a Strain Isolated from Bovine Mastitis

    PubMed Central

    Gazzola, Simona; Fontana, Cecilia; Bassi, Daniela; Cocconcelli, Pier-Sandro; von Wright, Atte

    2015-01-01

    The genome sequence of Lactococcus lactis subsp. cremoris Mast36, isolated from bovine mastitis, is reported here. This strain was shown to be able to grow in milk and still possess genes of vegetable origin. The genome also contains a cluster of genes associated with pathogenicity. PMID:25999570

  8. Effects of eliminating pyruvate node pathways and of coexpression of heterogeneous carboxylation enzymes on succinate production by Enterobacter aerogenes.

    PubMed

    Tajima, Yoshinori; Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

    2015-02-01

    Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. PMID:25416770

  9. Close genetic linkage of the determinants of the ribitol and D-arabitol catabolic pathways in Klebsiella aerogenes.

    PubMed

    Charnetzky, W T; Mortlock, R P

    1974-07-01

    Klebsiella aerogenes strain W70 has separate inducible pathways for the degradation of the pentitols ribitol and d-arabitol. These pathways are closely linked genetically as determined by transduction with phage PW52. There are two regulatory sites for the ribitol catabolic pathway as defined by loci for mutations to constitutive synthesis of ribitol dehydrogenase and d-ribulokinase, rbtB and rbtC. The two control sites are separated by a site represented by the dalB22 mutation. This mutation deprives the cell of the ability to induce synthesis of d-arabitol dehydrogenase and d-xylulokinase activities. Two additional regulatory mutations for the d-arabitol pathway, dalC31 and dalC37, map to the opposite side of rbtB13 relative to dalB22. The order of the genetic sites thus far determined for this region is dalK-dalD-dalC31, dalC37-rbtB13-dalB22-rbtC14-rbtD-rbtK, where dalK and dalD represent structural genes for the kinase and dehydrogenase of the d-arabitol pathway, respectively, and rbtK and rbtD represent the corresponding genes for the ribitol pathway. The two mutations that lead to constitutive synthesis of the d-arabitol-induced enzymes, dalC31 and dalC37, have different phenotypes with regard to their response to xylitol. The growth of dalC31 is inhibited by xylitol, but the toxicity can be reduced by increasing the levels of ribitol dehydrogenase either by induction with ribitol or by selection of a ribitol dehydrogenase-constitutive mutation.

  10. In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple beta-lactamases.

    PubMed

    Chen, Ya-Gang; Zhang, Ying; Yu, Yun-Song; Qu, Ting-Ting; Wei, Ze-Qing; Shen, Ping; Li, Lan-Juan

    2008-10-01

    Four clinical strains of extended-spectrum beta-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C(1) and C(2) were isolated prior to carbapenem therapy, whilst isolates C(3) and C(4) were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). beta-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C(1) in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple beta-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C(1) and C(2), isolates C(3) and C(4) failed to express OmpE36 owing to insertional inactivation by an IS903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene (kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C(3) and C(4) were apparently derived from the previously imipenem-susceptible isolates C(1) and C(2). Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.

  11. In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem.

    PubMed

    Philippe, Nadège; Maigre, Laure; Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

    2015-01-01

    Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen.

  12. Electronic structure analysis of the dinuclear metal center in the bioremediator glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

    PubMed

    Hadler, Kieran S; Mitić, Natasa; Yip, Sylvia Hsu-Chen; Gahan, Lawrence R; Ollis, David L; Schenk, Gerhard; Larrabee, James A

    2010-03-15

    The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous, dinuclear metallohydrolase that has potential application in the remediation of organophosphate nerve agents and pesticides. GpdQ employs an unusual reaction mechanism in which the enzyme is predominantly mononuclear in the resting state, and substrate binding induces the formation of the catalytically competent dinuclear center (Hadler et al. J. Am. Chem. Soc. 2008, 130, 14129). Reactivity is further modulated by the coordination flexibility of Asn80, a ligand that binds to the second, loosely bound metal ion (Hadler et al. J. Am. Chem. Soc. 2009, 131, 11900). It is proposed that hydrolysis is initiated by a terminal, metal-bound hydroxide molecule which is activated at unusually low pH by electrostatic/hydrogen bonding interactions with a bridging hydroxide species. In this study, electronic structure analysis of the dinuclear center is employed to study the coordination environment of the dinuclear center at the resting and product-bound stage of catalysis. This is achieved through the use of variable temperature, variable field magnetic circular dichroism experiments involving the Co(II)-substituted wild type enzyme and its Asn80Asp variant. The data support the above model for the catalytic mechanism whereby the metal ion-bridging hydroxide molecule activates a terminally bound hydroxide nucleophile. Replacement of Asn80 by an aspartate residue does prevent coordination flexibility but also leads to cleavage of the mu-hydroxide bridge and reduced reactivity. This is the first study to investigate the electronic structure of an enzyme with a mu-1,1-carboxylate bridged dicobalt(II) center.

  13. Effects of eliminating pyruvate node pathways and of coexpression of heterogeneous carboxylation enzymes on succinate production by Enterobacter aerogenes.

    PubMed

    Tajima, Yoshinori; Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

    2015-02-01

    Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.

  14. Detection of extended-spectrum beta-lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes.

    PubMed

    Tzelepi, E; Giakkoupi, P; Sofianou, D; Loukova, V; Kemeroglou, A; Tsakris, A

    2000-02-01

    The aim of the present study was to investigate the frequency of extended-spectrum beta-lactamases (ESBLs) in a consecutive collection of clinical isolates of Enterobacter spp. The abilities of various screening methods to detect ESBLs in enterobacters were simultaneously tested. Among the 68 consecutive isolates (56 Enterobacter cloacae and 12 Enterobacter aerogenes isolates) that were analyzed for beta-lactamase content, 21 (25 and 58%, respectively) possessed transferable ESBLs with pIs of 8.2 and phenotypic characteristics of SHV-type enzymes, 8 (14.3%) of the E. cloacae isolates produced a previously nondescribed, clavulanate-susceptible ESBL that exhibited a pI of 6.9 and that conferred a ceftazidime resistance phenotype on Escherichia coli transconjugants, and 2 E. cloacae isolates produced both of these enzymes. Among the total of 31 isolates that were considered ESBL producers, the Vitek ESBL detection test was positive for 2 (6.5%) strains, and the conventional double-disk synergy test (DDST) with amoxicillin-clavulanate and with expanded-spectrum cephalosporins and aztreonam was positive for 5 (16%) strains. Modifications of the DDST consisting of closer application of the disks (at 20 instead of 30 mm), the use of cefepime, and the use of both modifications increased the sensitivity of this test to 71, 61, and 90%, respectively. Of the 37 isolates for which isoelectric focusing failed to determine ESBLs, the Vitek test was false positive for 1 isolate and the various forms of DDSTs were false-positive for 3 isolates.

  15. Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

    PubMed Central

    Yamamoto, Yoko; Fukui, Keita; Nishio, Yousuke; Hashiguchi, Kenichi; Usuda, Yoshihiro; Sode, Koji

    2014-01-01

    Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production. PMID:25416770

  16. In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem

    PubMed Central

    Santini, Sébastien; Pinet, Elizabeth; Claverie, Jean-Michel; Davin-Régli, Anne-Véronique; Pagès, Jean-Marie; Masi, Muriel

    2015-01-01

    Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen. PMID:26398358

  17. Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis.

    PubMed Central

    de Ruyter, P G; Kuipers, O P; Beerthuyzen, M M; van Alen-Boerrigter, I; de Vos, W M

    1996-01-01

    The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG 1363, which is a nisin-transposon- and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control. PMID:8655538

  18. An extended dynamic model of Lactococcus lactis metabolism for mannitol and 2,3-butanediol production.

    PubMed

    Costa, Rafael S; Hartmann, Andras; Gaspar, Paula; Neves, Ana R; Vinga, Susana

    2014-03-01

    Biomedical research and biotechnological production are greatly benefiting from the results provided by the development of dynamic models of microbial metabolism. Although several kinetic models of Lactococcus lactis (a Lactic Acid Bacterium (LAB) commonly used in the dairy industry) have been developed so far, most of them are simplified and focus only on specific metabolic pathways. Therefore, the application of mathematical models in the design of an engineering strategy for the production of industrially important products by L. lactis has been very limited. In this work, we extend the existing kinetic model of L. lactis central metabolism to include industrially relevant production pathways such as mannitol and 2,3-butanediol. In this way, we expect to study the dynamics of metabolite production and make predictive simulations in L. lactis. We used a system of ordinary differential equations (ODEs) with approximate Michaelis-Menten-like kinetics for each reaction, where the parameters were estimated from multivariate time-series metabolite concentrations obtained by our team through in vivo Nuclear Magnetic Resonance (NMR). The results show that the model captures observed transient dynamics when validated under a wide range of experimental conditions. Furthermore, we analyzed the model using global perturbations, which corroborate experimental evidence about metabolic responses upon enzymatic changes. These include that mannitol production is very sensitive to lactate dehydrogenase (LDH) in the wild type (W.T.) strain, and to mannitol phosphoenolpyruvate: a phosphotransferase system (PTS(Mtl)) in a LDH mutant strain. LDH reduction has also a positive control on 2,3-butanediol levels. Furthermore, it was found that overproduction of mannitol-1-phosphate dehydrogenase (MPD) in a LDH/PTS(Mtl) deficient strain can increase the mannitol levels. The results show that this model has prediction capability over new experimental conditions and offers promising

  19. Quantifying the effect of hand wash duration, soap use, ground beef debris, and drying methods on the removal of Enterobacter aerogenes on hands.

    PubMed

    Jensen, Dane A; Danyluk, Michelle D; Harris, Linda J; Schaffner, Donald W

    2015-04-01

    Hand washing is recognized as a crucial step in preventing foodborne disease transmission by mitigating crosscontamination among hands, surfaces, and foods. This research was undertaken to establish the importance of several keys factors (soap, soil, time, and drying method) in reducing microorganisms during hand washing. A nonpathogenic nalidixic acid-resistant Enterobacter aerogenes surrogate for Salmonella was used to assess the efficacy of using soap or no soap for 5 or 20 s on hands with or without ground beef debris and drying with paper towel or air. Each experiment consisted of 20 replicates, each from a different individual with ∼ 6 log CFU/ml E. aerogenes on their hands. A reduction of 1.0 ± 0.4 and 1.7 ± 0.8 log CFU of E. aerogenes was observed for a 5-s wash with no soap and a 20-s wash with soap, respectively. When there was no debris on the hands, there was no significant difference between washing with and without soap for 20 s (P > 0.05). Likewise, there was no significant difference in the reductions achieved when washing without soap, whether or not debris was on the hands (P > 0.05). A significantly greater reduction (P < 0.05) in E. aerogenes (0.5 log CFU greater reduction) was observed with soap when there was ground beef debris on the hands. The greatest difference (1.1 log CFU greater average reduction) in effectiveness occurred when ground beef debris was on the hands and a 20-s wash with water was compared with a 20-s wash with soap. Significantly greater (P < 0.05) reductions were observed with paper towel drying compared with air (0.5 log CFU greater reductions). Used paper towels may contain high bacterial levels (>4.0 log CFU per towel) when hands are highly contaminated. Our results support future quantitative microbial risk assessments needed to effectively manage risks of foodborne illness in which food workers' hands are a primary cause.

  20. Quantifying the effect of hand wash duration, soap use, ground beef debris, and drying methods on the removal of Enterobacter aerogenes on hands.

    PubMed

    Jensen, Dane A; Danyluk, Michelle D; Harris, Linda J; Schaffner, Donald W

    2015-04-01

    Hand washing is recognized as a crucial step in preventing foodborne disease transmission by mitigating crosscontamination among hands, surfaces, and foods. This research was undertaken to establish the importance of several keys factors (soap, soil, time, and drying method) in reducing microorganisms during hand washing. A nonpathogenic nalidixic acid-resistant Enterobacter aerogenes surrogate for Salmonella was used to assess the efficacy of using soap or no soap for 5 or 20 s on hands with or without ground beef debris and drying with paper towel or air. Each experiment consisted of 20 replicates, each from a different individual with ∼ 6 log CFU/ml E. aerogenes on their hands. A reduction of 1.0 ± 0.4 and 1.7 ± 0.8 log CFU of E. aerogenes was observed for a 5-s wash with no soap and a 20-s wash with soap, respectively. When there was no debris on the hands, there was no significant difference between washing with and without soap for 20 s (P > 0.05). Likewise, there was no significant difference in the reductions achieved when washing without soap, whether or not debris was on the hands (P > 0.05). A significantly greater reduction (P < 0.05) in E. aerogenes (0.5 log CFU greater reduction) was observed with soap when there was ground beef debris on the hands. The greatest difference (1.1 log CFU greater average reduction) in effectiveness occurred when ground beef debris was on the hands and a 20-s wash with water was compared with a 20-s wash with soap. Significantly greater (P < 0.05) reductions were observed with paper towel drying compared with air (0.5 log CFU greater reductions). Used paper towels may contain high bacterial levels (>4.0 log CFU per towel) when hands are highly contaminated. Our results support future quantitative microbial risk assessments needed to effectively manage risks of foodborne illness in which food workers' hands are a primary cause. PMID:25836392

  1. Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium

    PubMed Central

    Audet, Pascal; Paquin, Celine; Lacroix, Christophe

    1989-01-01

    Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented. PMID:16347822

  2. Promoter and signal sequence from filamentous fungus can drive recombinant protein production in the yeast Kluyveromyces lactis.

    PubMed

    Madhavan, Aravind; Sukumaran, Rajeev K

    2014-08-01

    Cross-recognition of promoters from filamentous fungi in yeast can have important consequences towards developing fungal expression systems, especially for the rapid evaluation of their efficacy. A truncated 510bp inducible Trichoderma reesei cellobiohydrolase I (cbh1) promoter was tested for the expression of green fluorescent protein (GFP) in Kluyveromyces lactis after disrupting its native β-galactosidase (lac4) promoter. The efficiency of the CBH1 secretion signal was also evaluated by fusing it to the lac4 promoter of the yeast, which significantly increased the secretion of recombinant protein in K. lactis compared to the native α-mating factor secretion signal. The fungal promoter is demonstrated to have potential to drive heterologous protein production in K. lactis; and the small sized T. reesei cbh1 secretion signal can mediate the protein secretion in K. lactis with high efficiency. PMID:24661814

  3. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications. PMID:25447786

  4. Reduced Binding of the Endolysin LysTP712 to Lactococcus lactis ΔftsH Contributes to Phage Resistance.

    PubMed

    Roces, Clara; Campelo, Ana B; Escobedo, Susana; Wegmann, Udo; García, Pilar; Rodríguez, Ana; Martínez, Beatriz

    2016-01-01

    Absence of the membrane protease FtsH in Lactococcus lactis hinders release of the bacteriophage TP712. In this work we have analyzed the mechanism responsible for the non-lytic phenotype of L. lactis ΔftsH after phage infection. The lytic cassette of TP712 contains a putative antiholin-pinholin system and a modular endolysin (LysTP712). Inducible expression of the holin gene demonstrated the presence of a dual start motif which is functional in both wildtype and L. lactis ΔftsH cells. Moreover, simulating holin activity with ionophores accelerated lysis of wildtype cells but not L. lactis ΔftsH cells, suggesting inhibition of the endolysin rather than a role of FtsH in holin activation. However, zymograms revealed the synthesis of an active endolysin in both wildtype and L. lactis ΔftsH TP712 lysogens. A reporter protein was generated by fusing the cell wall binding domain of LysTP712 to the fluorescent mCherry protein. Binding of this reporter protein took place at the septa of both wildtype and L. lactis ΔftsH cells as shown by fluorescence microscopy. Nonetheless, fluorescence spectroscopy demonstrated that mutant cells bound 40% less protein. In conclusion, the non-lytic phenotype of L. lactis ΔftsH is not due to direct action of the FtsH protease on the phage lytic proteins but rather to a putative function of FtsH in modulating the architecture of the L. lactis cell envelope that results in a lower affinity of the phage endolysin to its substrate. PMID:26904011

  5. Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

    PubMed Central

    Gunge, N; Tamaru, A; Ozawa, F; Sakaguchi, K

    1981-01-01

    Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer. Images PMID:6257636

  6. Genome‐scale diversity and niche adaptation analysis of Lactococcus lactis by comparative genome hybridization using multi‐strain arrays

    PubMed Central

    Siezen, Roland J.; Bayjanov, Jumamurat R.; Felis, Giovanna E.; van der Sijde, Marijke R.; Starrenburg, Marjo; Molenaar, Douwe; Wels, Michiel; van Hijum, Sacha A. F. T.; van Hylckama Vlieg, Johan E. T.

    2011-01-01

    Summary Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non‐dairy niches, such as fermented plant material. Recently, these non‐dairy strains have gained increasing interest, as they have been described to possess flavour‐forming activities that are rarely found in dairy isolates and have diverse metabolic properties. We performed an extensive whole‐genome diversity analysis on 39 L. lactis strains, isolated from dairy and plant sources. Comparative genome hybridization analysis with multi‐strain microarrays was used to assess presence or absence of genes and gene clusters in these strains, relative to all L. lactis sequences in public databases, whereby chromosomal and plasmid‐encoded genes were computationally analysed separately. Nearly 3900 chromosomal orthologous groups (chrOGs) were defined on basis of four sequenced chromosomes of L. lactis strains (IL1403, KF147, SK11, MG1363). Of these, 1268 chrOGs are present in at least 35 strains and represent the presently known core genome of L. lactis, and 72 chrOGs appear to be unique for L. lactis. Nearly 600 and 400 chrOGs were found to be specific for either the subspecies lactis or subspecies cremoris respectively. Strain variability was found in presence or absence of gene clusters related to growth on plant substrates, such as genes involved in the consumption of arabinose, xylan, α‐galactosides and galacturonate. Further niche‐specific differences were found in gene clusters for exopolysaccharides biosynthesis, stress response (iron transport, osmotolerance) and bacterial defence mechanisms (nisin biosynthesis). Strain variability of functions encoded on known plasmids included proteolysis, lactose fermentation, citrate uptake, metal ion resistance and exopolysaccharides biosynthesis. The present study supports the view of L. lactis as a species with a very flexible

  7. Reduced Binding of the Endolysin LysTP712 to Lactococcus lactis ΔftsH Contributes to Phage Resistance

    PubMed Central

    Roces, Clara; Campelo, Ana B.; Escobedo, Susana; Wegmann, Udo; García, Pilar; Rodríguez, Ana; Martínez, Beatriz

    2016-01-01

    Absence of the membrane protease FtsH in Lactococcus lactis hinders release of the bacteriophage TP712. In this work we have analyzed the mechanism responsible for the non-lytic phenotype of L. lactis ΔftsH after phage infection. The lytic cassette of TP712 contains a putative antiholin–pinholin system and a modular endolysin (LysTP712). Inducible expression of the holin gene demonstrated the presence of a dual start motif which is functional in both wildtype and L. lactis ΔftsH cells. Moreover, simulating holin activity with ionophores accelerated lysis of wildtype cells but not L. lactis ΔftsH cells, suggesting inhibition of the endolysin rather than a role of FtsH in holin activation. However, zymograms revealed the synthesis of an active endolysin in both wildtype and L. lactis ΔftsH TP712 lysogens. A reporter protein was generated by fusing the cell wall binding domain of LysTP712 to the fluorescent mCherry protein. Binding of this reporter protein took place at the septa of both wildtype and L. lactis ΔftsH cells as shown by fluorescence microscopy. Nonetheless, fluorescence spectroscopy demonstrated that mutant cells bound 40% less protein. In conclusion, the non-lytic phenotype of L. lactis ΔftsH is not due to direct action of the FtsH protease on the phage lytic proteins but rather to a putative function of FtsH in modulating the architecture of the L. lactis cell envelope that results in a lower affinity of the phage endolysin to its substrate. PMID:26904011

  8. Caractéristiques bactériologiques des infections de liquide de dialyse péritonéale

    PubMed Central

    Jellouli, Manel; Ferjani, Meriem; Abidi, Kamel; Hammi, Yosra; Abdallah, Taieb Ben; Gargah, Tahar

    2015-01-01

    La péritonite infectieuse (PI) est la principale complication de la dialyse péritonéale (DP). L'objectif de notre travail était de déterminer l’écologie bactérienne des PI et d'adapter l'antibiothérapie selon les germes isolés et les résistances observées. Étude rétrospective effectuée chez tous les enfants traités par DP et ayant présenté une PI dans le service de pédiatrie de l'hôpital Charles Nicolle de Tunis (2004-2013). Au total, 61 ont développé 97 PI. L'incidence des PI était de 0,75 épisode/patient-année. La culture du LDP était négative dans 40 cas. Les Gram Positif ont été notés dans 56% des cas avec prédominance du Staphylococcus aureus. Les Gram négatif étaient retrouvés en seconde position (40%) représentés principalement par le Klebsiella pneumoniae et le Pseudomonas aeruginosa. Des souches de Staphylocoque méticilline résistant étaient isolées dans 21,4%. Les bactéries à Gram positif étaient résistantes aux céphalosporines de première génération dans 25% des cas et aucune résistance à la vancomycine n'avait été décelée. Les bactéries à Gram négatif avaient une résistance globale de 38% avec des souches β-lactamase à spectre élargi (BLSE). L'antibiothérapie empirique devra couvrir les germes à Gram positif par la vancomycine et les germes à Gram négatif par la ceftazidime. PMID:26893796

  9. Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis

    PubMed Central

    Dong, Xiangrong; Tian, Bing; Dai, Shang; Li, Tao; Guo, Linna; Tan, Zhongfang; Jiao, Zhen; Jin, Qingsheng; Wang, Yanping; Hua, Yuejin

    2015-01-01

    PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production. PMID:26562776

  10. Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis.

    PubMed

    Dong, Xiangrong; Tian, Bing; Dai, Shang; Li, Tao; Guo, Linna; Tan, Zhongfang; Jiao, Zhen; Jin, Qingsheng; Wang, Yanping; Hua, Yuejin

    2015-01-01

    PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production. PMID:26562776

  11. Generation of Lactococcus lactis capable of coexpressing epidermal growth factor and trefoil factor to enhance in vitro wound healing.

    PubMed

    Huynh, Evanna; Li, Julang

    2015-06-01

    Epidermal growth factor (EGF) and trefoil factor 3 (TFF3) are peptides that actively support the restitution and repair of mucosal epithelial barriers. Previous studies have shown that TFF3 enhanced EGF effect in wound healing, suggesting that the combined application of the two factors may be advantageous in clinical tissue repair. Expression of multiple proteins in a single host is a desirable approach in a biotechnological process, allowing to reduce cost and increase production efficiency. The aim of the present study was to study the feasibility of coexpressing EGF and TFF3 in food grade bacteria, Lactococcus lactis (L. lactis). Using an expression construct allowing simultaneous translation of two separate recombinant peptides, we generated a L. lactis that coexpressed and secreted EGF and TFF3 dually (LL-ET). Western blot analysis revealed that LL-ET secreted 45-54 % more total recombinant peptides (EGF+TFF3) per flask fermentation and 21-37 % more total recombinant proteins in bioreactor fermentation compared to their single factor expressing L. lactis counterparts (LL-EGF and LL-TFF3, respectively). The resulted recombinant EGF and TFF3 showed enhancement in wound healing activity in vitro. Our data suggest that the dual expression and secretion of EGF and TFF3 by L. lactis effectively accelerated cell migration, demonstrating potential future oral application of L. lactis fermentation product containing dual factors or a cocktail of factors to potentially treat intestinal damage and inflammation.

  12. Expression of PprI from Deinococcus radiodurans Improves Lactic Acid Production and Stress Tolerance in Lactococcus lactis.

    PubMed

    Dong, Xiangrong; Tian, Bing; Dai, Shang; Li, Tao; Guo, Linna; Tan, Zhongfang; Jiao, Zhen; Jin, Qingsheng; Wang, Yanping; Hua, Yuejin

    2015-01-01

    PprI is a general switch protein that regulates the expression of certain proteins involved in pathways of cellular resistance in the extremophilic bacterium Deinococcus radiodurans. In this study, we transformed pprI into Lactococcus lactis strain MG1363 using the lactococcal shuttle vector pMG36e and investigated its effects on the tolerance and lactic acid production of L. lactis while under stress. PprI was stably expressed in L. lactis as confirmed by western blot assays. L. lactis expressing PprI exhibited significantly improved resistance to oxidative stress and high osmotic pressure. This enhanced cellular tolerance to stressors might be due to the regulation of resistance-related genes (e.g., recA, recO, sodA, and nah) by pprI. Moreover, transformed L. lactis demonstrated increased lactic acid production, attributed to enhanced lactate dehydrogenase activity. These results suggest that pprI can improve the tolerance of L. lactis to environmental stresses, and this transformed bacterial strain is a promising candidate for industrial applications of lactic acid production.

  13. A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.

    PubMed

    Médici, Rosario; Garaycoechea, Juan I; Valino, Ana L; Pereira, Claudio A; Lewkowicz, Elizabeth S; Iribarren, Adolfo M

    2014-04-01

    Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

  14. Susceptibility of the multi-drug resistant strain of Enterobacter aerogenes EA289 to the terpene alcohols from Cistus ladaniferus essential oil.

    PubMed

    Guinoiseau, Elodie; Lorenzi, Vannina; Luciani, Anne; Tomi, Félix; Casanova, Joseph; Berti, Liliane

    2011-08-01

    The essential oil (EO) of Cistus ladaniferus was separated into non polar, moderately polar and polar fractions by column chromatography. The EO and its fractions were analysed by gas chromatography in combination with retention indices [GC-(RI)] and 13C nuclear magnetic resonance (13C NMR) spectroscopy. A minimum inhibitory concentration (MIC) assay was used to evaluate their antibacterial activity against Gram-positive and Gram-negative pathogens of clinical relevance, including a multi-drug resistant (MDR) strain. The most polar fraction, constituted by mono- and sesquiterpene alcohols, strongly inhibited the growth of all tested bacteria with MIC values ranging from 0.05 to 0.8 mg/mL. More importantly, this fraction displayed high activity against the MDR strain of Enterobacter aerogenes EA289. Transmission electron microscopy (TEM) observations of the MDR bacteria treated with the terpene alcohol-rich fraction revealed cell wall distortion with an outer cytoplasmic membrane detachment. The susceptibility of the MDR strain of E. aerogenes EA289 to the polar fraction of C. ladaniferus oil suggests the possible use of these natural products to treat infections caused by highly resistant bacteria.

  15. Genetic diversity and mycotoxin production of Fusarium lactis species complex isolates from sweet pepper.

    PubMed

    Van Poucke, Kris; Monbaliu, Sofie; Munaut, Françoise; Heungens, Kurt; De Saeger, Sarah; Van Hove, François

    2012-02-01

    An internal fruit rot disease of sweet peppers was first detected in Belgium in 2003. Research conducted mostly in Canada indicates that this disease is primarily caused by Fusarium lactis Pirotta. Ninety-eight Fusarium isolates obtained from diseased sweet peppers from Belgium, as well as from other countries (Canada, the Netherlands and the United Kingdom) were identified by sequencing the translation elongation factor 1α (EF). Of these 98 isolates, 13 were identified as F. oxysporum Schltdl., nine as F. proliferatum (Matsush.) Nirenberg and two belonged to clade 3 of the F. solani species complex. Of the 74 remaining isolates, the EF sequence showed 97% to 98% similarity to F. lactis. Of these isolates, the β-tubulin (TUB), calmodulin (CAM) and the second largest subunit of RNA polymerase II (RPB2) genes were also sequenced. Analysis of the combined sequences revealed that the 74 isolates share nine combined sequences that correspond to nine multilocus sequence types (STs), while the F. lactis neotype strain and one other strain, both isolated from figs, form a separate ST. Together, these 10 STs represent a monophyletic F. lactis species complex (FLASC). An unusually high level of genetic diversity was observed between (groups of) these STs. Two of them (ST5 and ST6) fulfilled the criteria for species recognition based on genealogical exclusivity and together represent a new monophyletic species lineage (FLASC-1). The seven other STs, together with the F. lactis neotype ST, form a paraphyletic species lineage in the African clade of the Gibberella fujikuroi species complex (GFSC). From each of the 10 STs, the mycotoxin production was assessed using a multi-mycotoxin liquid chromatography mass spectrometry method. Out of the 27 analyzed mycotoxins, beauvericin and fumonisins were detected in sweet pepper tissue and in maize kernels. The 10 STs clearly differed in the amount of mycotoxin produced, but there was only limited congruence between the production

  16. Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase

    PubMed Central

    Rodríguez, Ángel Pereira; Leiro, Rafael Fernández; Trillo, M Cristina; Cerdán, M Esperanza; Siso, M Isabel González; Becerra, Manuel

    2006-01-01

    Background The β-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing. The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process. Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the β-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization. Results The highest levels of intracellular and extracellular β-galactosidase were obtained when the segment corresponding to the five domain of K. lactis β-galactosidase was replaced by the corresponding five domain of the A. niger β-galactosidase. Taking into account that this replacement may affect other parameters related to the activity or the stability of the hybrid protein, a thoroughly study was performed. Both pH (6.5) and temperature (40°C) for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the β-galactosidase of K. lactis and, unlike this, the activity of the hybrid protein was increased by the presence of Ni2+. The affinity for

  17. Genetic diversity and mycotoxin production of Fusarium lactis species complex isolates from sweet pepper.

    PubMed

    Van Poucke, Kris; Monbaliu, Sofie; Munaut, Françoise; Heungens, Kurt; De Saeger, Sarah; Van Hove, François

    2012-02-01

    An internal fruit rot disease of sweet peppers was first detected in Belgium in 2003. Research conducted mostly in Canada indicates that this disease is primarily caused by Fusarium lactis Pirotta. Ninety-eight Fusarium isolates obtained from diseased sweet peppers from Belgium, as well as from other countries (Canada, the Netherlands and the United Kingdom) were identified by sequencing the translation elongation factor 1α (EF). Of these 98 isolates, 13 were identified as F. oxysporum Schltdl., nine as F. proliferatum (Matsush.) Nirenberg and two belonged to clade 3 of the F. solani species complex. Of the 74 remaining isolates, the EF sequence showed 97% to 98% similarity to F. lactis. Of these isolates, the β-tubulin (TUB), calmodulin (CAM) and the second largest subunit of RNA polymerase II (RPB2) genes were also sequenced. Analysis of the combined sequences revealed that the 74 isolates share nine combined sequences that correspond to nine multilocus sequence types (STs), while the F. lactis neotype strain and one other strain, both isolated from figs, form a separate ST. Together, these 10 STs represent a monophyletic F. lactis species complex (FLASC). An unusually high level of genetic diversity was observed between (groups of) these STs. Two of them (ST5 and ST6) fulfilled the criteria for species recognition based on genealogical exclusivity and together represent a new monophyletic species lineage (FLASC-1). The seven other STs, together with the F. lactis neotype ST, form a paraphyletic species lineage in the African clade of the Gibberella fujikuroi species complex (GFSC). From each of the 10 STs, the mycotoxin production was assessed using a multi-mycotoxin liquid chromatography mass spectrometry method. Out of the 27 analyzed mycotoxins, beauvericin and fumonisins were detected in sweet pepper tissue and in maize kernels. The 10 STs clearly differed in the amount of mycotoxin produced, but there was only limited congruence between the production

  18. Plasmids of Raw Milk Cheese Isolate Lactococcus lactis subsp. lactis Biovar diacetylactis DPC3901 Suggest a Plant-Based Origin for the Strain ▿ †

    PubMed Central

    Fallico, Vincenzo; McAuliffe, Olivia; Fitzgerald, Gerald F.; Ross, R. Paul

    2011-01-01

    The four-plasmid complement of the raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for Lactococcus, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (orf11 and orf25). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (orf19 to orf21 [orf19-21]), which has a single database homolog on a plant-derived Leuconostoc plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with orf19-21 of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na+ and K+ transport. The presence of numerous plant-associated markers in L. lactis DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for Lactococcus strains. PMID:21803914

  19. Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study.

    PubMed

    Merenstein, Daniel J; Tan, Tina P; Molokin, Aleksey; Smith, Keisha Herbin; Roberts, Robert F; Shara, Nawar M; Mete, Mihriye; Sanders, Mary Ellen; Solano-Aguilar, Gloria

    2015-01-01

    Probiotics are live microorganisms that, when administered in sufficient doses, provide health benefits on the host. The United States Food and Drug Administration (FDA) requires phase I safety studies for probiotics when the intended use of the product is as a drug. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp lactis (B. lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of adults who were prescribed a 10-day course of antibiotics for a respiratory infection. Secondary aims were to assess the ability of BB-12 to affect the expression of whole blood immune markers associated with cell activation and inflammatory response. A phase I, double-blinded, randomized controlled study was conducted in compliance with FDA guidelines for an Investigational New Drug (IND). Forty participants were randomly assigned to consume 4 ounces of either BB-12 -supplemented yogurt or non-supplemented control yogurt daily for 10 d. The primary outcome was to assess safety and tolerability, assessed by the number of reported adverse events. A total of 165 non-serious adverse events were reported, with no differences between the control and BB-12 groups. When compared to the control group, B lactis fecal levels were modestly higher in the BB-12-supplemented group. In a small subset of patients, changes in whole blood expression of genes associated with regulation and activation of immune cells were detected in the BB-12-supplemented group. BB-12-supplemented yogurt is safe and well tolerated when consumed by healthy adults concurrently taking antibiotics. This study will form the basis for future randomized clinical trials investigating the potential immunomodulatory effects of BB-12-supplemented yogurt in a variety of disease states.

  20. Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study

    PubMed Central

    Merenstein, Daniel J; Tan, Tina P; Molokin, Aleksey; Smith, Keisha Herbin; Roberts, Robert F; Shara, Nawar M; Mete, Mihriye; Sanders, Mary Ellen; Solano-Aguilar, Gloria

    2015-01-01

    Probiotics are live microorganisms that, when administered in sufficient doses, provide health benefits on the host. The United States Food and Drug Administration (FDA) requires phase I safety studies for probiotics when the intended use of the product is as a drug. The purpose of the study was to determine the safety of Bifidobacterium animalis subsp lactis (B. lactis) strain BB-12 (BB-12)-supplemented yogurt when consumed by a generally healthy group of adults who were prescribed a 10-day course of antibiotics for a respiratory infection. Secondary aims were to assess the ability of BB-12 to affect the expression of whole blood immune markers associated with cell activation and inflammatory response. A phase I, double-blinded, randomized controlled study was conducted in compliance with FDA guidelines for an Investigational New Drug (IND). Forty participants were randomly assigned to consume 4 ounces of either BB-12 -supplemented yogurt or non-supplemented control yogurt daily for 10 d. The primary outcome was to assess safety and tolerability, assessed by the number of reported adverse events. A total of 165 non-serious adverse events were reported, with no differences between the control and BB-12 groups. When compared to the control group, B lactis fecal levels were modestly higher in the BB-12-supplemented group. In a small subset of patients, changes in whole blood expression of genes associated with regulation and activation of immune cells were detected in the BB-12-supplemented group. BB-12-supplemented yogurt is safe and well tolerated when consumed by healthy adults concurrently taking antibiotics. This study will form the basis for future randomized clinical trials investigating the potential immunomodulatory effects of BB-12-supplemented yogurt in a variety of disease states. PMID:25569274

  1. Méningo-vascularite bactérienne révélant un adénome hypophysaire

    PubMed Central

    Bouchal, Siham; Razzouki, Hanane; Elkhyat, Salma Ibn; Maaroufi, Mustafa; El Ouali, Ouarda; Messouak, Ouafae; Belahsen, Mohammed Faouzi

    2015-01-01

    Un macroadénome à prolactine se présente généralement par un syndrome hormonal associé à un syndrome tumoral. Une méningite bactérienne compliquant un macroprolactinome en dehors de toute thérapie médicale ou chirurgicale est rarement rapporté dans la littérature. Mme R.C âgée de 48 ans s'est présentée aux urgences pour trouble de conscience fébrile. La ponction lombaire a révélé une méningite bactérienne. L'imagerie cérébrale a mis en évidence un aspect de vascularite cérébrale et un processus de la loge sellaire avec lyse du plancher faisant évoquer une brèche ostéoméningée. Le bilan biologique a montré une hyperprolactinémie à 200 mg/dl. La patiente est mise sous antibiothérapie à dose méningée et une corticothérapie associée à un traitement par la Cabergoline. Le traitement chirurgical de la brèche ostéoméningée s'est fait par voie endonasale. L’évolution est marquée par une nette amélioration clinique et biologique et l'absence de récidive de la méningite après un recul de 14 mois. Un macroprolactinome peut provoquer une brèche ostéoméningée en dehors de tout traitement médical ou chirurgical et avoir comme première manifestation une méningite infectieuse. PMID:25995804

  2. Defined bacterial culture development for methane generation from lactose. [Streptococcus lactis; Clostridium formicoaceticum; Methanococcus mazei

    SciTech Connect

    Yang, S.T.; Tang, I.C.; Okos, M.R.

    1988-06-20

    The defined microbial cultures for methane generation from lactose were investigated. A mixed culture consisting of homolactic (Streptococcus lactis), homoacetic (Clostridium formicoaceticum), and acetate-utilizing methanogenic (Methanococcus mazei) bacteria was used to convert lactose and whey permeate to methane at mesophilic temperatures (35-37/sup 0/C) and a pH around 7.0. Lactose was first converted to lactic acid by S. lactis, then to acetic acid by C. formicoaceticum, and finally to methane and CO/sub 2/ by M. mazei. About 5.3 mol methane were obtained from each mole of lactose consumed, and the conversion of acetate to methane was the rate-limiting step for this mixed-culture fermentation.

  3. Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

    PubMed

    Loera-Arias, María J; Villatoro-Hernández, Julio; Parga-Castillo, Miguel A; Salcido-Montenegro, Alejandro; Barboza-Quintana, Oralia; Muñoz-Maldonado, Gerardo E; Montes-de-Oca-Luna, Roberto; Saucedo-Cárdenas, Odila

    2014-12-01

    Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications.

  4. Fractionation of Dipeptidase Activities of Streptococcus lactis and Dipeptidase Specificity of Some Lactic Acid Bacteria

    PubMed Central

    Sørhaug, Terje; Solberg, Peter

    1973-01-01

    Proteins in sonic extracts of Streptococcus lactis were separated by starch-gel electrophoresis at high voltage. Each slab was sliced longitudinally, and half was stained for peptidases in a mixture containing a peptide, L-amino acid oxidase (snake venom), peroxidase, and o-dianisdine; the other half was stained in amido black for protein. In addition to sonic treatment, trypsin also released enzyme from acetone-treated cells. Glycyl-L-phenylalanine, L-phenylalanyl-glycine, L-alanyl-L-phenylalanine, and L-phenylalanyl-L-alanine served as substrates in characterizing the enzymes. Five different fractions of various specificities appeared in the gels. Broad-range substrate specificities were found for sonic extracts of S. lactis, S. cremoris, S. durans, and Lactobacillus acidophilus. Images PMID:4633426

  5. Secretion of biologically active human interleukin 22 (IL-22) by Lactococcus lactis.

    PubMed

    Loera-Arias, María J; Villatoro-Hernández, Julio; Parga-Castillo, Miguel A; Salcido-Montenegro, Alejandro; Barboza-Quintana, Oralia; Muñoz-Maldonado, Gerardo E; Montes-de-Oca-Luna, Roberto; Saucedo-Cárdenas, Odila

    2014-12-01

    Interleukin-22 (IL-22) participates in the modulation of innate immunity and inflammation. This cytokine has important therapeutic potential, such as with ulcerative colitis, liver and lung injury, and infection, in different animal models. We generated a Lactococcus lactis strain that secretes human IL-22 under the regulation of the nisin-inducible promoter. Identification and secretion of this cytokine was demonstrated using western blots of culture supernatants from IL-22-expressing bacteria. The recombinant IL-22 protein produced by L. lactis was biologically active as determined by its ability to induce IL-10 secretion when co-cultured with a colon epithelial cell line in vitro. We consider this novel strain a promising live vaccine for various therapeutic applications. PMID:25214209

  6. Construction and application of a food-grade expression system for Lactococcus lactis.

    PubMed

    Lu, Wenwei; Kong, Jian; Kong, Wentao

    2013-06-01

    A food-grade host/vector expression system for Lactococcus lactis was constructed using alanine racemase gene (alr) as the complementation marker. We obtained an alanine racemase auxotrophic mutant L. lactis NZ9000Δalr by double-crossover recombination using temperature-sensitive integration plasmid pG(+)host9 and a food-grade vector pALR with entirely lactococcal DNA elements, including lactococcal replicon, nisin-inducible promoter PnisA and the alr gene from Lactobacillus casei BL23 as a complementation marker. By using the new food-grade host/vector system, the green fluorescent protein and capsid protein of porcine circovirus type II were successfully overexpressed under the nisin induction. These results indicate that this food-grade host/vector expression system has application potential as an excellent antigen delivery vehicle, and is also suitable for the use in the manufacture of ingredients for the food industry. PMID:22674186

  7. The Pasteur effect and catabolite repression in an oxidative yeast, Kluyveromyces lactis.

    PubMed

    Royt, P W; MacQuillan, A M

    1979-01-01

    The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation. Extracts from these cells contained ATP-sensitive phosphofructokinase activity. Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation. Phosphofructokinase in these cells was ATP-insensitive. The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation. These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K. lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase. Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity. The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts.

  8. Physiological adaptation of the bacterium Lactococcus lactis in response to the production of human CFTR.

    PubMed

    Steen, Anton; Wiederhold, Elena; Gandhi, Tejas; Breitling, Rainer; Slotboom, Dirk Jan

    2011-07-01

    Biochemical and biophysical characterization of CFTR (the cystic fibrosis transmembrane conductance regulator) is thwarted by difficulties to obtain sufficient quantities of correctly folded and functional protein. Here we have produced human CFTR in the prokaryotic expression host Lactococcus lactis. The full-length protein was detected in the membrane of the bacterium, but the yields were too low (< 0.1% of membrane proteins) for in vitro functional and structural characterization, and induction of the expression of CFTR resulted in growth arrest. We used isobaric tagging for relative and absolute quantitation based quantitative proteomics to find out why production of CFTR in L. lactis was problematic. Protein abundances in membrane and soluble fractions were monitored as a function of induction time, both in CFTR expression cells and in control cells that did not express CFTR. Eight hundred and forty six proteins were identified and quantified (35% of the predicted proteome), including 163 integral membrane proteins. Expression of CFTR resulted in an increase in abundance of stress-related proteins (e.g. heat-shock and cell envelope stress), indicating the presence of misfolded proteins in the membrane. In contrast to the reported consequences of membrane protein overexpression in Escherichia coli, there were no indications that the membrane protein insertion machinery (Sec) became overloaded upon CFTR production in L. lactis. Nutrients and ATP became limiting in the control cells as the culture entered the late exponential and stationary growth phases but this did not happen in the CFTR expressing cells, which had stopped growing upon induction. The different stress responses elicited in E. coli and L. lactis upon membrane protein production indicate that different strategies are needed to overcome low expression yields and toxicity.

  9. Genotype-phenotype matching analysis of 38 Lactococcus lactis strains using random forest methods

    PubMed Central

    2013-01-01

    Background Lactococcus lactis is used in dairy food fermentation and for the efficient production of industrially relevant enzymes. The genome content and different phenotypes have been determined for multiple L. lactis strains in order to understand intra-species genotype and phenotype diversity and annotate gene functions. In this study, we identified relations between gene presence and a collection of 207 phenotypes across 38 L. lactis strains of dairy and plant origin. Gene occurrence and phenotype data were used in an iterative gene selection procedure, based on the Random Forest algorithm, to identify genotype-phenotype relations. Results A total of 1388 gene-phenotype relations were found, of which some confirmed known gene-phenotype relations, such as the importance of arabinose utilization genes only for strains of plant origin. We also identified a gene cluster related to growth on melibiose, a plant disaccharide; this cluster is present only in melibiose-positive strains and can be used as a genetic marker in trait improvement. Additionally, several novel gene-phenotype relations were uncovered, for instance, genes related to arsenite resistance or arginine metabolism. Conclusions Our results indicate that genotype-phenotype matching by integrating large data sets provides the possibility to identify gene-phenotype relations, possibly improve gene function annotation and identified relations can be used for screening bacterial culture collections for desired phenotypes. In addition to all gene-phenotype relations, we also provide coherent phenotype data for 38 Lactococcus strains assessed in 207 different phenotyping experiments, which to our knowledge is the largest to date for the Lactococcus lactis species. PMID:23530958

  10. Acetate Utilization in Lactococcus lactis Deficient in Lactate Dehydrogenase: a Rescue Pathway for Maintaining Redox Balance

    PubMed Central

    Hols, Pascal; Ramos, Ana; Hugenholtz, Jeroen; Delcour, Jean; de Vos, Willem M.; Santos, Helena; Kleerebezem, Michiel

    1999-01-01

    Acetate was shown to improve glucose fermentation in Lactococcus lactis deficient in lactate dehydrogenase. 13C and 1H nuclear magnetic resonance studies using [2-13C]glucose and [2-13C]acetate as substrates demonstrated that acetate was exclusively converted to ethanol. This novel pathway provides an alternative route for NAD+ regeneration in the absence of lactate dehydrogenase. PMID:10464231

  11. Use of Lactococcus lactis to enrich sourdough bread with γ-aminobutyric acid.

    PubMed

    Bhanwar, Seema; Bamnia, Meenakshi; Ghosh, Moushumi; Ganguli, Abhijit

    2013-02-01

    Fried sourdough bread (bhatura) with an elevated amount of γ-aminobutyric acid (GABA) was produced using lactic acid bacteria (LAB). The LAB starter was screened and isolated from pickled yam showing highest GABA content and was identified as Lactococcus lactis subsp. lactis. The maximum GABA production in de Man Rogosa Sharpe (MRS) media supplemented with monosodium glutamate (MSG) was 110 mg/100 ml at pH 5, and 1-3% NaCl did not change the production of GABA significantly (p>0.05). When MSG was replaced with Vigna mungo in sourdough, the amount of GABA for bhatura was 226.22 mg/100 g representing about 10-fold increase. A sensory evaluation resulted as the overall general acceptability of bhatura to be 4.91 ± 0.03 on a five-point hedonic scale. Thus, the results indicated the potential of L. lactis as a LAB starter for the production of GABA-enriched bhatura. Although other physiological effects can be expected in the product, animal and clinical studies are mandatory prior to application of this food.

  12. Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system.

    PubMed

    Langella, P; Le Loir, Y

    1999-02-01

    Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitopeprotein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

  13. A Virulent Phage Infecting Lactococcus garvieae, with Homology to Lactococcus lactis Phages

    PubMed Central

    Eraclio, Giovanni; Tremblay, Denise M.; Lacelle-Côté, Alexia; Labrie, Simon J.; Fortina, Maria Grazia

    2015-01-01

    A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor. PMID:26407890

  14. Probiotic assessment of Enterococcus durans 6HL and Lactococcus lactis 2HL isolated from vaginal microflora.

    PubMed

    Nami, Yousef; Abdullah, Norhafizah; Haghshenas, Babak; Radiah, Dayang; Rosli, Rozita; Khosroushahi, Ahmad Yari

    2014-08-01

    Forty-five lactic acid bacteria (LAB) were isolated from the vaginal specimens of healthy fertile women, and the identities of the bacteria were confirmed by sequencing of their 16S rDNA genes. Among these bacteria, only four isolates were able to resist and survive in low pH, bile salts and simulated in vitro digestion conditions. Lactococcus lactis 2HL, Enterococcus durans 6HL, Lactobacillus acidophilus 36YL and Lactobacillus plantarum 5BL showed the best resistance to these conditions. These strains were evaluated further to assess their ability to adhere to human intestinal Caco-2 cells. Lactococcus lactis 2HL and E. durans 6HL were the most adherent strains. In vitro tests under neutralized pH proved the antimicrobial activity of both strains. Results revealed that the growth of Escherichia coli O26, Staphylococcus aureus and Shigella flexneri was suppressed by both LAB strains. The antibiotic susceptibility tests showed that these strains were sensitive to all nine antibiotics: vancomycin, tetracycline, ampicillin, penicillin, gentamicin, erythromycin, clindamycin, sulfamethoxazole and chloramphenicol. These data suggest that E. durans 6HL and Lactococcus lactis 2HL could be examined further for their useful properties and could be developed as new probiotics.

  15. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666.

    PubMed

    Del Rio, Beatriz; Linares, Daniel M; Redruello, Begoña; Martin, Maria Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2015-12-01

    Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC[1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2], which is also transcriptionally regulated by carbon catabolic repression (CCR) via glucose, but not by other sugars such as lactose and galactose [1], [3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR) [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE59514. PMID:26697381

  16. Biotechnological and safety characterization of Enterococcus lactis, a recently described species of dairy origin.

    PubMed

    Morandi, Stefano; Silvetti, Tiziana; Brasca, Milena

    2013-01-01

    The biotechnological and safety properties of a recently described enterococcal species, Enterococcus lactis, were investigated. With regard to the technological properties, in milk all the strains tested had weak acidifying and proteolytic activities, generally medium reduction activity over 24 h (-102 mV < Eh < -2 mV) and low lipolytic activity on tributyrin agar. The isolates were tested for resistance against 14 antibiotics and none of the studied strains were classified as resistant to clinically important antibiotics such as ampicillin, erythromycin, penicillin G, tetracycline and vancomycin. Furthermore, PCR-based detection did not identify any of the common genetic determinants for vancomycin, tetracycline and erythromycin resistance. The E. lactis strains showed good survival in simulated in vitro digestion and were able to inhibit the growth of Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Clostridium sporogenes, Clostridium tyrobutyricum and Pseudomonas syringae. Screening for enterocin structural genes showed that all isolates harboured the entP gene. The presence of nine virulence factor genes (cylA, asa1, gelE, hyl, esp, ace, efaA, hdc and tdc) was investigated by PCR and no virulence determinants were detected. This study highlights that the recently described E. lactis may be a potential source of novel strains with interesting features that could be used for fermented dairy foods.

  17. Biotechnological and safety characterization of Enterococcus lactis, a recently described species of dairy origin.

    PubMed

    Morandi, Stefano; Silvetti, Tiziana; Brasca, Milena

    2013-01-01

    The biotechnological and safety properties of a recently described enterococcal species, Enterococcus lactis, were investigated. With regard to the technological properties, in milk all the strains tested had weak acidifying and proteolytic activities, generally medium reduction activity over 24 h (-102 mV < Eh < -2 mV) and low lipolytic activity on tributyrin agar. The isolates were tested for resistance against 14 antibiotics and none of the studied strains were classified as resistant to clinically important antibiotics such as ampicillin, erythromycin, penicillin G, tetracycline and vancomycin. Furthermore, PCR-based detection did not identify any of the common genetic determinants for vancomycin, tetracycline and erythromycin resistance. The E. lactis strains showed good survival in simulated in vitro digestion and were able to inhibit the growth of Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Clostridium sporogenes, Clostridium tyrobutyricum and Pseudomonas syringae. Screening for enterocin structural genes showed that all isolates harboured the entP gene. The presence of nine virulence factor genes (cylA, asa1, gelE, hyl, esp, ace, efaA, hdc and tdc) was investigated by PCR and no virulence determinants were detected. This study highlights that the recently described E. lactis may be a potential source of novel strains with interesting features that could be used for fermented dairy foods. PMID:22961639

  18. Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis.

    PubMed

    Yu, L; O'Sullivan, D J

    2014-02-01

    Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85 °C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.

  19. Recyclable Strategy for the Production of High-Purity Galacto-oligosaccharides by Kluyveromyces lactis.

    PubMed

    Sun, Huaisheng; You, Shengping; Wang, Mengfan; Qi, Wei; Su, Rongxin; He, Zhimin

    2016-07-20

    A recyclable strategy for the production of high-purity (>95%) galacto-oligosaccharides (GOS) was developed using Kluyveromyces lactis in both the synthesis and purification steps. For the synthesis of GOS, ethanol-permeabilized cells (p-cells) of K. lactis were used because the enhanced permeability facilitated the mass transfer of the substrate and the release of oligosaccharide products. For the purification of GOS, non-permeabilized K. lactis cells (np-cells) were preferred as a result of their intrinsic cell membrane barrier toward GOS, which led to the selective consumption of carbohydrate. In this way, undesired glucose, galactose, and lactose in the raw GOS solution can be completely removed. This strategy is recyclable not only because of the high stability and reusability of p-cells and np-cells but also because the ethanol, which is simultaneously generated during the purification, can be reused for the preparation of p-cells. The strategy proposed in this study is a promising candidate for the efficient production of high-purity GOS.

  20. The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

    PubMed Central

    Castelain, Mickaël; Duviau, Marie-Pierre; Canette, Alexis; Schmitz, Philippe; Loubière, Pascal; Cocaign-Bousquet, Muriel; Piard, Jean-Christophe; Mercier-Bonin, Muriel

    2016-01-01

    Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis. PMID:27010408

  1. Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F44

    PubMed Central

    Zhang, Jian; Caiyin, Qinggele; Feng, Wenjing; Zhao, Xiuli; Qiao, Bin; Zhao, Guangrong; Qiao, Jianjun

    2016-01-01

    Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process. However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. lactis F44, respectively. Comparing to the 2810 IU/mL nisin yield of the original strain F44, the nisin titer of the engineered strains over-expressing hdeAB, ldh and murG, increased to 3850, 3979 and 4377 IU/mL, respectively. These engineered strains showed more stable intracellular pH value during the fermentation process. Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. Co-overexpression of hdeAB, murG, and ldh(Z) in strain F44 resulted in the nisin titer of 4913 IU/mL. The engineered strain (ABGL) could grow on plates with pH 4.2, comparing to the surviving pH 4.6 of strain F44. The fed-batch fermentation showed nisin titer of the co-expression L. lactis strain could reach 5563 IU/mL with lower pH condition and longer cultivation time. This work provides a novel strategy of constructing robust strains for use in industry process. PMID:27306587

  2. Genome-Wide Transcriptional Responses to Carbon Starvation in Nongrowing Lactococcus lactis

    PubMed Central

    Ercan, Onur; Wels, Michiel; Smid, Eddy J.

    2015-01-01

    This paper describes the transcriptional adaptations of nongrowing, retentostat cultures of Lactococcus lactis to starvation. Near-zero-growth cultures (μ = 0.0001 h−1) obtained by extended retentostat cultivation were exposed to starvation by termination of the medium supply for 24 h, followed by a recovery period of another 24 h by reinitiating the medium supply to the retentostat culture. During starvation, the viability of the culture was largely retained, and the expression of genes involved in transcription and translational machineries, cell division, and cell membrane energy metabolism was strongly repressed. Expression of these genes was largely recovered following the reinitiation of the medium supply. Starvation triggered the elevated expression of genes associated with synthesis of branched-chain amino acids, histidine, purine, and riboflavin. The expression of these biosynthesis genes was found to remain at an elevated level after reinitiation of the medium supply. In addition, starvation induced the complete gene set predicted to be involved in natural competence in L. lactis KF147, and the elevated expression of these genes was sustained during the subsequent recovery period, but our attempts to experimentally demonstrate natural transformation in these cells failed. Mining the starvation response gene set identified a conserved cis-acting element that resembles the lactococcal CodY motif in the upstream regions of genes associated with transcription and translational machineries, purine biosynthesis, and natural transformation in L. lactis, suggesting a role for CodY in the observed transcriptome adaptations to starvation in nongrowing cells. PMID:25636846

  3. Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis.

    PubMed

    Masuda, Masaki; Freguia, Stefano; Wang, Yung-Fu; Tsujimura, Seiya; Kano, Kenji

    2010-06-01

    Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around -0.4V vs. Ag|AgCl. Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor. High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. lactis to exploit exogenous flavins for anodic glucose oxidation was confirmed by tests where flavin-type compounds were supplied to the bacterium in well defined media. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide (FMN) was also exploited by L. lactis as a redox shuttle, unlike flavin adenine dinucleotide (FAD), possibly due to the absence of a specific transporter for the latter. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results.

  4. Measuring Kinetic Dissociation/Association Constants Between Lactococcus lactis Bacteria and Mucins Using Living Cell Probes

    PubMed Central

    Le, Doan Thanh Lam; Guérardel, Yann; Loubière, Pascal; Mercier-Bonin, Muriel; Dague, Etienne

    2011-01-01

    In this work we focused on quantifying adhesion between Lactococcus lactis, the model for lactic acid bacteria (LAB) and mucins. Interactions between two strains of L. lactis (IBB477 and MG1820 as control) and pig gastric mucin–based coating were measured and compared with the use of atomic force microscopy. Analysis of retraction force-distance curves shed light on the differential contributions of nonspecific and specific forces. An increased proportion of specific adhesive events was obtained for IBB477 (20% vs. 5% for the control). Blocking assays with free pig gastric mucin and its O-glycan moiety showed that oligosaccharides play a major (but not exclusive) role in L. lactis-mucins interactions. Specific interactions were analyzed in terms of kinetic constants. An increase in the loading rate of atomic force microscope tip led to a higher force between interacting biological entities, which was directly linked to the kinetic dissociation constant (Koff). Enhancing the contact time between the tip and the sample allowed an increase in the interaction probability, which can be related to the kinetic association constant (Kon). Variations in the loading rate and contact time enabled us to determine Kon (3.3 × 102 M−1·s−1) and Koff (0.46 s−1), and the latter was consistent with values given in the literature for sugar-protein interactions. PMID:22261074

  5. A new and efficient phosphate starvation inducible expression system for Lactococcus lactis.

    PubMed

    Sirén, Noora; Salonen, Kalle; Leisola, Matti; Nyyssölä, Antti

    2008-07-01

    A new expression system for Lactococcus lactis was developed. The system is based on a phosphate starvation inducible pstF promoter of L. lactis MG1363. Intracellular beta-galactosidase and secreted alpha-amylase were produced using this tightly regulated system. No evidence of regulatory sites in regions of the 5'-end of the pstF coding sequence was found. High expression levels of the beta-galactosidase gene were obtained using the original pstF RBS in a phosphate-depleted medium. The results suggested that with the phosphate starvation inducible system, it is possible to achieve expression levels comparable to the ones obtained with the widely used nisin-controlled gene expression system (NICE). A specific beta-galactosidase activity of 670 microkat g(-1) using a phosphate-depleted medium and an alpha-amylase activity of 3.6 microkat l(-1) in a bioreactor cultivation were produced. The advantages of the current expression system include that no prior removal of phosphate from the medium in bioreactor scale is required, and no additions of inducing agents are needed. Furthermore, the system can be operated in L. lactis without introduction of regulatory genes into the host.

  6. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666

    PubMed Central

    del Rio, Beatriz; Linares, Daniel M.; Redruello, Begoña; Martin, Maria Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P.; Ladero, Victor; Alvarez, Miguel A.

    2015-01-01

    Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC[1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2], which is also transcriptionally regulated by carbon catabolic repression (CCR) via glucose, but not by other sugars such as lactose and galactose [1], [3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR) [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE59514. PMID:26697381

  7. Construction and expression of food-grade β-galactosidase gene in Lactococcus Lactis.

    PubMed

    Zhang, Wen; Wang, Chuan; Huang, Chengyu; Yu, Qian; Liu, Hengchuan; Zhang, Chaowu; Pei, Xiaofang

    2011-02-01

    Recombinant Lactococcus lactis MG1363/pMG36e-lacZ exhibiting high β-galactosidase activities were constructed by us in the previous study. However, erythromycin resistance present in these recombinants restricted their practical application in food preparation. This study was conducted to delete the gene coding for erythromycin resistance present in recombinant L. lactis, as a result of which these bacteria express food-grade β-galactosidase. In this study, the recombinant plasmid pMG36e-lacZ was digested with restriction enzymes BclI and HpaI and the food-grade plasmid FGZW was rebuilt. FGZW was transformed into Escherichia coli JM109 and L. lactis MG1363. Erythromycin resistance, enzyme activity determination, gene sequencing and SDS-PAGE analysis indicated that these new recombinant bacteria lost erythromycin resistance and its relevant gene but still expressed β-galactosidase activities, although a decrease in the expression of β-galactosidase of these new strains was observed. The β-galactosidase food-grade expression system was successfully constructed and it could provide a new solution for the management of lactose intolerance. These results might promote the usage of gene-modified microorganisms and related technology in the food sector, which has the highest priority for food safety.

  8. Transcriptomic profile of aguR deletion mutant of Lactococcus lactis subsp. cremoris CECT 8666.

    PubMed

    Del Rio, Beatriz; Linares, Daniel M; Redruello, Begoña; Martin, Maria Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2015-12-01

    Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) is a dairy strain that catabolizes agmatine (a decarboxylated derivative of arginine) into the biogenic amine putrescine by the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are responsible for the deamination of agmatine to putrescine and are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC[1]. aguR encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2], which is also transcriptionally regulated by carbon catabolic repression (CCR) via glucose, but not by other sugars such as lactose and galactose [1], [3]. Here we report the transcriptional profiling of the aguR gene deletion mutant (L. lactis subsp. cremoris CECT 8666 ∆aguR) [2] compared to the wild type strain, both grown in M17 medium with galactose as carbon source and supplemented with agmatine. The transcriptional profiling data of AguR-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE59514.

  9. Successive emergence of extended-spectrum beta-lactamase-producing and carbapenemase-producing Enterobacter aerogenes isolates in a university hospital.

    PubMed

    Biendo, M; Canarelli, B; Thomas, D; Rousseau, F; Hamdad, F; Adjide, C; Laurans, G; Eb, F

    2008-03-01

    Sixty-two clinical isolates of Enterobacter aerogenes resistant to expanded-spectrum cephalosporins were collected between July 2003 and May 2005. Among these isolates, 23 (37.1%) were imipenem (IPM) susceptible, and 39 (62.9%) were IPM insusceptible, of which 89.7% (35/39) were resistant and 10.3% (4/39) were intermediate. Isolate genotypes were compared by pulsed-field gel electrophoresis. Of 62 isolates, 48 belonged to epidemic pulsotype A (77.4%). This pulsotype included 37.5% and 58.4% of beta-lactam phenotypes b and a, respectively. Nine isolates (14.5%) belonged to pulsotype E, which included 22.3% and 77.7% of phenotypes b and a, respectively. The beta-lactamases with pIs of 5.4, 6.5, 8.2, and 8.2 corresponded to extended-spectrum beta-lactamases (ESBLs) TEM-20, TEM-24, SHV-5, and SHV-12, respectively. Of 39 IPM-insusceptible E. aerogenes isolates, 26 (66.6%) were determined to be metallo-beta-lactamase producers, by using a phenotypic method. Of these isolates, 24 harbored a bla(IMP-1) gene encoding a protein with a pI of >9.5, and two carried the bla(VIM-2) gene encoding a protein with a pI of 5.3, corresponding to beta-lactamases IMP-1 and VIM-2, respectively. The remaining 13 (33.4%) isolates were negative for the bla(IMP-1) and bla(VIM-2) genes but showed an alteration of their outer membrane proteins (OMPs). Ten of these isolates produced the two possible OMPs (32 and 42 kDa), with IPM MICs between 8 and 32 microg/ml, and three others produced only a 32-kDa OMP with IPM MICs >32 microg/ml. This work demonstrates that, in addition to resistance to expanded-spectrum cephalosporins, IPM resistance can occur in ESBL-producing E. aerogenes isolates by carbapenemase production or by the loss of porin in the outer membrane.

  10. A bacteriocin gene cluster able to enhance plasmid maintenance in Lactococcus lactis

    PubMed Central

    2014-01-01

    Background Lactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus. Results Attempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation. Conclusions Inserting the Lcn972 cluster into

  11. Recombinant porcine epidermal growth factor-secreting Lactococcus lactis promotes the growth performance of early-weaned piglets

    PubMed Central

    2014-01-01

    Background Epidermal growth factor (EGF) is an important growth factor in regulation of cell proliferation, differentiation, survival and apoptosis. Studies showed that food-grade Lactococcus lactis (L. lactis) and NICE expression system have superior performance in exogenous protein expression. This study aimed to construct and express porcine EGF (pEGF), and use L. lactis as vehicle for producing and delivering pEGF. Furthermore, investigating biological activity of pEGF and exploring applications feasibility of combination effects of L. lactis and pEGF on early weaned piglets’ production. Results A recombinant Lactococcus lactis which produced and secreted pEGF at 1000 ng/ml in culture supernatant was generated. Secreted pEGF was a fully biologically active protein, as demonstrated by its capacity to stimulate L929 mouse fibroblast cell line proliferation in vitro. For in vivo study, forty piglets were randomly allocated to control, antibiotic control, empty vector-expressing L. lactis (LL-EV) and pEGF-secreting L. lactis (LL-pEGF). After 14 d of rearing, final body weight and average daily gain in LL-pEGF were greater (P < 0.05, 8.95 vs. 8.37 kg, 206.1 vs. 157.7 g/day, respectively) than those in control, but no significant differences between LL-pEGF, LL-EV and antibiotic control. Overall period average daily feed intake was higher in LL-pEGF, LL-EV and antibiotic control than in control (P < 0.05, 252.9, 255.6, 250.0, 207.3 g/day, respectively). No significant difference was observed on ADFI/ADG. LL-pEGF increased villous height in the duodenum, jejunum and ileum than in control and LL-EV (P < 0.05). Sucrase in the 3 intestinal segments, aminopeptidase A in the duodenum and Jejunum, aminopeptidase N and dipeptidase IV in the duodenum in LL-pEGF were higher than those in control (P < 0.05). Furthermore, Escherichia coli and Enterococcus counts decreased in the ileum and Lactobacillus increased in the ileum and cecum digesta in LL-pEGF compare with the

  12. Induction of specific immune responses in piglets by intramuscular immunization with fimbrial adhesin FaeG expressed in Lactococcus lactis.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei

    2013-08-01

    Fimbrial adhesin plays a critical role in the pathogenesis of enterotoxigenic Escherichia coli (ETEC)-induced piglet diarrhoea. Lactococcus lactis is an attractive food-grade host for the production of heterologous antigens. We previously demonstrated that fimbrial adhesin FaeG was expressed in L. lactis and that oral immunization in mice with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses. In the present study, we explored the immune responses of piglets induced by intramuscular vaccination with recombinant L. lactis expressing rFaeG. Intramuscular vaccination resulted in significantly elevated serum IgG level and modest increases in serum IgA and IgM levels. In addition, IgG, IgA, and IgM antibody secreting cells were induced in the spleen, mesenteric lymph nodes, and jejunum. The growth performance of piglets was not influenced by intramuscular vaccination. The results suggest that L. lactis expressing FaeG is a promising candidate vaccine against ETEC. PMID:23540979

  13. Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71.

    PubMed

    Varma, Nadimpalli Ravi S; Toosa, Haryanti; Foo, Hooi Ling; Alitheen, Noorjahan Banu Mohamed; Nor Shamsudin, Mariana; Arbab, Ali S; Yusoff, Khatijah; Abdul Rahim, Raha

    2013-01-01

    In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle.

  14. Use of murine models to detect the allergenicity of genetically modified Lactococcus lactis NZ9000/pNZPNK.

    PubMed

    Chiang, Shen-Shih; Liu, Chin-Feng; Ku, Ting-Wei; Mau, Jeng-Leun; Lin, Hsin-Tang; Pan, Tzu-Ming

    2011-04-27

    By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.

  15. Subcutaneous or oral immunization of mice with Lactococcus lactis expressing F4 fimbrial adhesin FaeG.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 (K88) fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZ8112-faeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis. In this work, BALB/c mice were immunized with recombinant L. lactis to further determine the immunogenicity of recombinant FaeG (rFaeG) via the subcutaneous or oral route. Subcutaneous immunization in mice with recombinant L. lactis induced a significant increase in the F4-specific serum IgG titer and the number of antibody-secreting cells (ASCs) in the spleen. Oral immunization of mice with recombinant L. lactis induced mucosal and systemic F4-specific immune responses and increased the number of ASCs in the spleen, mesenteric lymph nodes and Peyer's patches. High-dose (2.8 × 10(11) CFU) recombinant strains and adjuvant cholera toxin B subunit enhanced specific mucosal immune responses. The results suggest the feasibility of delivering rFaeG expressed in L. lactis to the immune system in order to induce an F4-specific immune response. PMID:23386358

  16. The rhizome of the multidrug-resistant Enterobacter aerogenes genome reveals how new "killer bugs" are created because of a sympatric lifestyle.

    PubMed

    Diene, Seydina M; Merhej, Vicky; Henry, Mireille; El Filali, Adil; Roux, Véronique; Robert, Catherine; Azza, Saïd; Gavory, Frederick; Barbe, Valérie; La Scola, Bernard; Raoult, Didier; Rolain, Jean-Marc

    2013-02-01

    Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of

  17. The rhizome of the multidrug-resistant Enterobacter aerogenes genome reveals how new "killer bugs" are created because of a sympatric lifestyle.

    PubMed

    Diene, Seydina M; Merhej, Vicky; Henry, Mireille; El Filali, Adil; Roux, Véronique; Robert, Catherine; Azza, Saïd; Gavory, Frederick; Barbe, Valérie; La Scola, Bernard; Raoult, Didier; Rolain, Jean-Marc

    2013-02-01

    Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of

  18. Characterization of KPC-2-producing Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Enterobacter aerogenes, and Klebsiella oxytoca isolates from a Chinese Hospital.

    PubMed

    Luo, Yanping; Yang, Jiyong; Ye, Liyan; Guo, Lin; Zhao, Qiang; Chen, Rong; Chen, Yong; Han, Xuelin; Zhao, Jingya; Tian, Shuguang; Han, Li

    2014-08-01

    Twelve nonduplicated KPC-2-producing enterobacterial isolates, including three Escherichia coli, two Citrobacter freundii, two Enterobacter cloacae, four Enterobacter aerogenes, and one Klebsiella oxytoca, were collected from various clinical samples within 18 months (March 2011 to September 2012). Two of the 12 patients died from infections caused by KPC-2-producing pathogens, while the rest of the patients with KPC-2-producing pathogens improved or were cured. The majority of the clinical isolates exhibited a high-level of resistance to oxyimino-cephalosporins and carbapenems, and possessed self-transferable bla(KPC-2)-carrying plasmids with sizes ranging from 20 to 120 kb. Most isolates carried bla(CTX-M) and plasmid-mediated quinolone resistance genes, while some isolates produced 16S rRNA methylases (ArmA or RmtB). The genetic environment of bla(KPC-2) of most clinical strains was consistent with the genetic structure surrounding bla(KPC-2) on the plasmid pKP048, which contains an integration structure of a Tn3-based transposon and partial Tn4401 segment. Inserted fragments (truncated bla(TEM)) were detected upstream of the bla(KPC-2) gene for two E. aerogenes strains. In conclusion, the enterobacterial isolates exhibited sporadic emergence and did not arise by clonal spread at our hospital. The outcome of infections caused by KPC-producing enterobacterial isolates and their mortality were closely associated with the baseline condition of patients. The spread of bla(KPC-2) gene between different enterobacterial species in China was mainly mediated by horizontal transfer of the Tn3-based transposons and not the bla(KPC-2)-carrying plasmids.

  19. Parameter Estimation of Actuators for Benchmark Active Control Technology (BACT) Wind Tunnel Model with Analysis of Wear and Aerodynamic Loading Effects

    NASA Technical Reports Server (NTRS)

    Waszak, Martin R.; Fung, Jimmy

    1998-01-01

    This report describes the development of transfer function models for the trailing-edge and upper and lower spoiler actuators of the Benchmark Active Control Technology (BACT) wind tunnel model for application to control system analysis and design. A simple nonlinear least-squares parameter estimation approach is applied to determine transfer function parameters from frequency response data. Unconstrained quasi-Newton minimization of weighted frequency response error was employed to estimate the transfer function parameters. An analysis of the behavior of the actuators over time to assess the effects of wear and aerodynamic load by using the transfer function models is also presented. The frequency responses indicate consistent actuator behavior throughout the wind tunnel test and only slight degradation in effectiveness due to aerodynamic hinge loading. The resulting actuator models have been used in design, analysis, and simulation of controllers for the BACT to successfully suppress flutter over a wide range of conditions.

  20. Performance of the BacT Alert 3D System Versus Solid Media for Recovery and Drug Susceptibility Testing of Mycobacterium tuberculosis in a Tertiary Hospital in Korea

    PubMed Central

    Kim, Seoung-Cheol; Kim, Jin-Sook; Choi, In Hwan; Kim, Jiro; Woo, Jeongim; Kim, Soojin; Lee, Hyeong Woo; Sezim, Monoldorova; Cho, Sang-Nae

    2016-01-01

    Background Tuberculosis (TB) is a major health problem, and accurate and rapid diagnosis of multidrug-resistant (MDR) and extended drug-resistant (XDR) TB is important for appropriate treatment. In this study, performances of solid and liquid culture methods were compared with respect to MDR- and XDR-TB isolate recovery and drug susceptibility testing. Methods Sputum specimens from 304 patients were stained with Ziehl-Neelsen method. Mycobacterium tuberculosis (Mtb) isolates were tested for recovery on Löwenstein-Jensen (LJ) medium and the BacT Alert 3D system. For drug susceptibility testing of Mtb, isolates were evaluated on M-KIT plates and the BacT Alert 3D system. Results The recovery rates were 94.9% (206/217) and 98.2% (213/217) for LJ medium and the BacT Alert 3D system, respectively (kappa coefficient, 0.884). The rate of drug resistance was 13.4% for at least one or more drugs, 6.0% for MDR-TB and 2.3% for XDR-TB. M-KIT plate and BacT 3D Alert 3D system were comparable in drug susceptibility testing for isoniazid (97.7%; kappa coefficient, 0.905) and rifampin (98.6%; kappa coefficient, 0.907). Antibiotic resistance was observed using M-KIT plates for 24 of the total 29 Mtb isolates (82.8%). Conclusion The liquid culture system showed greater reduction in the culture period, as compared with LJ medium; however, drug susceptibility testing using M-KIT plates was advantageous for simultaneous testing against multiple drug targets. PMID:27790280

  1. Bacteriocin-Producing Lactic Acid Bacteria Isolated from Mangrove Forests in Southern Thailand as Potential Bio-Control Agents: Purification and Characterization of Bacteriocin Produced by Lactococcus lactis subsp. lactis KT2W2L.

    PubMed

    Hwanhlem, Noraphat; Biscola, Vanessa; El-Ghaish, Shady; Jaffrès, Emmanuel; Dousset, Xavier; Haertlé, Thomas; H-Kittikun, Aran; Chobert, Jean-Marc

    2013-12-01

    The aim of this work was to purify and characterize the bacteriocin produced by Lactococcus lactis subsp. lactis KT2W2L previously isolated from mangrove forests in southern Thailand, in order to evaluate its potential as new food protective agent. The active peptide from the cell-free supernatant of this strain was purified in 4 steps: (1) precipitation with 70 % saturated ammonium sulfate, (2) elution on a reversed-phase cartridge using different concentrations of acetonitrile, (3) cation-exchange chromatography and (4) final purification by reversed-phase HPLC on a C8 column. The molecular mass of 3,329.5254 Da of the purified bacteriocin, determined by mass spectrometry, is nearly identical to that of peptide nisin Z. The activity of the purified bacteriocin was unaffected by pH (2.0-10.0), thermostable but was sensitive to proteolytic enzymes. The bacteriocin activity was stable after 8 weeks of storage at -20 °C and 7 weeks of storage at 4 °C, but decreased after 3 weeks of storage at 37 °C. It was stable when incubated for 1 month at 4 °C in 0-30 % NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activity against similar bacterial strains, food-spoilage and food-borne pathogens. L. lactis subsp. lactis KT2W2L was sensitive to kanamycin, penicillin and tetracycline but resistant to ampicillin, gentamicin and vancomycin. The fragment obtained after amplification of genomic DNA from L. lactis subsp. lactis KT2W2L, with specific primers for bacteriocin genes, presented 99 % homology to the nisin Z gene. PCR amplification demonstrated that L. lactis subsp. lactis KT2W2L does not harbor virulence genes cylA, cylB, efaAfs and esp. The bacteriocin and its producing strain may find application as bio-preservatives for reduction in food-spoilage and food-borne pathogens in food products.

  2. Genetically Engineered Lactococcus lactis Protect against House Dust Mite Allergy in a BALB/c Mouse Model

    PubMed Central

    Ai, Chunqing; Zhang, Qiuxiang; Ren, Chengcheng; Wang, Gang; Liu, Xiaoming; Tian, Fengwei; Zhao, Jianxin; Zhang, Hao; Chen, Yong Q.; Chen, Wei

    2014-01-01

    Background Mucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model. Methods Three strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall) were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro. Results Der p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes. Conclusion Oral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance. PMID:25290938

  3. Metabolome analysis of milk fermented by γ-aminobutyric acid-producing Lactococcus lactis.

    PubMed

    Hagi, Tatsuro; Kobayashi, Miho; Nomura, Masaru

    2016-02-01

    γ-Aminobutyric acid (GABA) is one of the most important functional components in fermented foods because of its physiological functions, such as neurotransmission and antihypertensive activities. However, little is known about components other than GABA in GABA-rich fermented foods. A metabolomic approach offers an opportunity to discover bioactive and flavor components in fermented food. To find specific components in milk fermented with GABA-producing Lactococcus lactis 01-7, we compared the components found in GABA-rich fermented milk with those found in control milk fermented without GABA production using capillary electrophoresis time-of-flight mass spectrometry. A principal component analysis score plot showed a clear differentiation between the control milk fermented with L. lactis 01-1, which does not produce GABA, and GABA-rich milk fermented with a combination of L. lactis strains 01-1 and 01-7. As expected, the amount of GABA in GABA-rich fermented milk was much higher (1,216-fold) than that of the control milk. Interestingly, the amount of Orn was also much higher (27-fold) than that of the control milk. Peptide analysis showed that levels of 6 putative angiotensin-I-converting enzyme (ACE)-inhibitory peptides were also higher in the GABA-rich fermented milk. Furthermore, ACE-inhibitory activity of GABA-rich fermented milk tended to be higher than that of the control milk. These results indicate that the GABA-producing strain 01-7 provides fermented milk with other functional components in addition to GABA.

  4. Transcriptome profiling of Lactococcus lactis subsp. cremoris CECT 8666 in response to agmatine.

    PubMed

    Del Rio, Beatriz; Redruello, Begoña; Martin, M Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2016-03-01

    The dairy strain Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC, which encodes the proteins necessary for agmatine uptake and its conversion into putrescine [1], [2]. The first gene of the cluster, aguR, encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2]. The catabolic operon aguBDAC is transcriptionally activated by agmatine [2] and transcriptionally regulated by carbon catabolite repression (CCR) via glucose, but not by other sugars such as lactose or galactose [1], [3]. On the contrary, the transcription of the aguR regulatory gene is not subject to CCR regulation [1], [3] nor is regulated by agmatine [2]. In this study we report the transcriptional profiling of L. lactis subsp. cremoris CECT 8666 grown in M17 medium with galactose (GalM17) as carbon source and supplemented with agmatine, compared to that of the strain grown in the same culture medium without agmatine. The transcriptional profiling data of agmatine-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under Accession no. GSE74808. PMID:26981381

  5. Microencapsulation of Bifidobacterium animalis subsp. lactis and Lactobacillus acidophilus in cocoa butter using spray chilling technology

    PubMed Central

    Pedroso, D.L.; Dogenski, M.; Thomazini, M.; Heinemann, R.J.B.; Favaro-Trindade, C.S.

    2013-01-01

    In the present study, the cells of Bifidobacterium animalis subsp. lactis (BI-01) and Lactobacillus acidophilus (LAC-04) were encapsulated in cocoa butter using spray-chilling technology. Survival assays were conducted to evaluate the resistance of the probiotics to the spray-chilling process, their resistance to the simulated gastric and intestinal fluids (SGF and SIF), and their stability during 90 days of storage. The viability of the cells was not affected by microencapsulation. The free and encapsulated cells of B. animalis subsp. lactis were resistant to both SGF and SIF. The micro-encapsulated cells of L. acidophilus were more resistant to SGF and SIF than the free cells; the viability of the encapsulated cells was enhanced by 67%, while the free cells reached the detection limit of the method (103 CFU/g). The encapsulated probiotics were unstable when they were stored at 20 °C. The population of encapsulated L. acidophilus decreased drastically when they were stored at 7 °C; only 20% of cells were viable after 90 days of storage. The percentage of viable cells of the encapsulated B. animalis subsp.lactis, however, was 72% after the same period of storage. Promising results were obtained when the microparticles were stored at −18 °C; the freeze granted 90 days of shelf life to the encapsulated cells. These results suggest that the spray-chilling process using cocoa butter as carrier protects L. acidophilus from gastrointestinal fluids. However, the viability of the cells during storage must be improved. PMID:24516445

  6. Hydrolysis of oligofructoses by the recombinant beta-fructofuranosidase from Bifidobacterium lactis.

    PubMed

    Janer, Carolina; Rohr, Lukas M; Peláez, Carmen; Laloi, Maryse; Cleusix, Valentine; Requena, Teresa; Meile, Leo

    2004-05-01

    The ability of the beta-fructofuranosidase (EC 3.2.1.26) from Bifidobacterium lactis DSM 10140T to cleave a variety of fructooligosaccharides was characterised. We identified its gene on a cloned chromosomal DNA fragment by sequence similarity (69% identity) to the putative CscA protein encoded in the Bifidobacterium longum genome. The deduced amino acid sequence of 532 residues (59.4 kDa) appeared to be identical to the beta-fructofuranosidase from the same strain recently described by Ehrmann et al. (Curr. Microbiol. 2003, 46, 391-397). However, the characterisation of the heterologously expressed enzyme showed several discrepancies to the referred study. First, the B. lactis beta-fructofuranosidase gene was found to have 41% identity with CscA from E. coli in contrast to the 16% reported, therefore it was assigned to as CscA protein instead of BfrA. Second, we observed only low activity of the enzyme towards sucrose (6%) instead of the 100% previously reported. Instead, we measured highest activity (100%) of the enzyme with the oligofructose Raftilose as a substrate compared with the inulin of low degree of polymerisation Raftiline LS (29%) and the highly polymerised Raftiline HP (10%). Altogether, the enzyme showed high affinity to terminal beta(2-1) glycosyl linkages between fructose moieties. The Km values obtained for Raftilose, Raftiline LS and sucrose were 0.12, 7.08 and 8.37 mM, respectively, and V(max) values for the conversion to fructose were calculated to be 5, 21 and 17 micromol/min per mg of protein, respectively. Growth of B. lactis was supported by fructans of low degree of polymerisation (Raftilose and Raftiline LS), whereas we observed no growth with highly polymerised inulin (Raftiline HP).

  7. Enzymatic synthesis and characterization of hydroquinone galactoside using Kluyveromyces lactis lactase.

    PubMed

    Kim, Go-Eun; Lee, Jin-Ha; Jung, Sun-Hwa; Seo, Eun-Seong; Jin, Sheng-De; Kim, Ghahyun J; Cha, Jaeho; Kim, Eui-Joong; Park, Ki-Deok; Kim, Doman

    2010-09-01

    Hydroquinone galactoside (HQ-Gal) as a potential skin whitening agent was synthesized by the reaction of lactase (beta-galactosidase) from Kluyveromyces lactis, Aspergillus oryzae, Bacillus circulans, and Thermus sp. with lactose as a donor and HQ as an acceptor. Among these lactases, the acceptor reaction involving HQ and lactose with K. lactis lactase showed a higher conversion ratio to HQ-Gal (60.27%). HQ-Gal was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HQ-Gal was determined by nuclear magnetic resonance, and the ionic product was observed at m/z 295 (C12H16O7Na)+ using matrix assisted laser desorption ionization time-of-flight mass spectrometry. HQ-Gal was identified as 4-hydroxyphenyl-beta-d-galactopyranoside. The optimum conditions for HQ-Gal synthesis by K. lactis determined using response surface methodology were 50 mM HQ, 60 mM lactose, and 20 U mL(-1) lactase. These conditions produced a yield of 2.01 g L(-1) HQ-Gal. The half maximal inhibitory concentration (IC50) of diphenylpicrylhydrazyl scavenging activity was 3.31 mM, indicating a similar antioxidant activity compared to beta-arbutin (IC50=3.95 mM). The Ki value of HQ-Gal (0.75 mM) against tyrosinase was smaller than that of beta-arbutin (Ki=1.97 mM), indicating its superiority as an inhibitor. HQ-Gal inhibited (23%) melanin synthesis without being significantly toxic to the cells, while beta-arbutin exhibited only 8% reduction of melanin synthesis in B16 melanoma cells compared with the control. These results indicate that HQ-Gal may be a suitable functional component in the cosmetics industry.

  8. Transcriptome profiling of Lactococcus lactis subsp. cremoris CECT 8666 in response to agmatine

    PubMed Central

    del Rio, Beatriz; Redruello, Begoña; Martin, M. Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P.; Ladero, Victor; Alvarez, Miguel A.

    2015-01-01

    The dairy strain Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC, which encodes the proteins necessary for agmatine uptake and its conversion into putrescine [1], [2]. The first gene of the cluster, aguR, encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2]. The catabolic operon aguBDAC is transcriptionally activated by agmatine [2] and transcriptionally regulated by carbon catabolite repression (CCR) via glucose, but not by other sugars such as lactose or galactose [1], [3]. On the contrary, the transcription of the aguR regulatory gene is not subject to CCR regulation [1], [3] nor is regulated by agmatine [2]. In this study we report the transcriptional profiling of L. lactis subsp. cremoris CECT 8666 grown in M17 medium with galactose (GalM17) as carbon source and supplemented with agmatine, compared to that of the strain grown in the same culture medium without agmatine. The transcriptional profiling data of agmatine-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under Accession no. GSE74808. PMID:26981381

  9. Mechanism of citrate metabolism by an oxaloacetate decarboxylase-deficient mutant of Lactococcus lactis IL1403.

    PubMed

    Pudlik, Agata M; Lolkema, Juke S

    2011-08-01

    Citrate metabolism in resting cells of Lactococcus lactis IL1403(pFL3) results in the formation of two end products from the intermediate pyruvate, acetoin and acetate (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:706-714, 2011). Pyruvate is formed from citrate following uptake by the transporter CitP through the subsequent actions of citrate lyase and oxaloacetate decarboxylase. The present study describes the metabolic response of L. lactis when oxaloacetate accumulates in the cytoplasm. The oxaloacetate decarboxylase-deficient mutant ILCitM(pFL3) showed nearly identical rates of citrate consumption, but the end product profile in the presence of glucose shifted from mainly acetoin to only acetate. In addition, in contrast to the parental strain, the mutant strain did not generate proton motive force. Citrate consumption by the mutant strain was coupled to the excretion of oxaloacetate, with a yield of 80 to 85%. Following citrate consumption, oxaloacetate was slowly taken up by the cells and converted to pyruvate by a cryptic decarboxylase and, subsequently, to acetate. The transport of oxaloacetate is catalyzed by CitP. The parental strain IL1403(pFL3) containing CitP consumed oxaloacetate, while the original strain, IL1403, not containing CitP, did not. Moreover, oxaloacetate consumption was enhanced in the presence of L-lactate, indicating exchange between oxaloacetate and L-lactate catalyzed by CitP. Hence, when oxaloacetate inadvertently accumulates in the cytoplasm, the physiological response of L. lactis is to excrete oxaloacetate in exchange with citrate by an electroneutral mechanism catalyzed by CitP. Subsequently, in a second step, oxaloacetate is taken up by CitP and metabolized to pyruvate and acetate.

  10. Transcriptome profiling of Lactococcus lactis subsp. cremoris CECT 8666 in response to agmatine.

    PubMed

    Del Rio, Beatriz; Redruello, Begoña; Martin, M Cruz; Fernandez, Maria; de Jong, Anne; Kuipers, Oscar P; Ladero, Victor; Alvarez, Miguel A

    2016-03-01

    The dairy strain Lactococcus lactis subsp. cremoris CECT 8666 (formerly GE2-14) synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway [1]. The AGDI cluster of L. lactis is composed by five genes aguR, aguB, aguD, aguA and aguC. The last four genes are co-transcribed as a single policistronic mRNA forming the catabolic operon aguBDAC, which encodes the proteins necessary for agmatine uptake and its conversion into putrescine [1], [2]. The first gene of the cluster, aguR, encodes a transmembrane protein that functions as a one-component signal transduction system that senses the agmatine concentration of the medium and accordingly regulates the transcription of aguBDAC[2]. The catabolic operon aguBDAC is transcriptionally activated by agmatine [2] and transcriptionally regulated by carbon catabolite repression (CCR) via glucose, but not by other sugars such as lactose or galactose [1], [3]. On the contrary, the transcription of the aguR regulatory gene is not subject to CCR regulation [1], [3] nor is regulated by agmatine [2]. In this study we report the transcriptional profiling of L. lactis subsp. cremoris CECT 8666 grown in M17 medium with galactose (GalM17) as carbon source and supplemented with agmatine, compared to that of the strain grown in the same culture medium without agmatine. The transcriptional profiling data of agmatine-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under Accession no. GSE74808.

  11. Enhancement of nisin production by Lactococcus lactis in periodically re-alkalized cultures.

    PubMed

    Guerra, Nelson Pérez; Castro, Lorenzo Pastrana

    2003-10-01

    Synthesis of nisin as well as biomass production by Lactococcus lactis subsp. lactis CECT (Colección Española de Cultivos Tipo) 539 on both hydrolysed mussel-processing waste and whey medium were followed in three fixed volume fed-batch fermentations, with re-alkalization cycles. The two cultures on mussel-processing waste (MPW) were fed with a 240 g/l concentrated glucose and with a concentrated MPW (about 100 g of glucose/l). The culture on whey was fed with a mixture of concentrated whey (48 g of total sugars/l) and a 400 g/l concentrated lactose. The three cultures were mainly characterized with higher nisin titres [49.7, 109.6 and 124.7 bacteriocin activity units (AU)/ml respectively] compared with the batch process on de Man, Rogosa and Sharpe [(1960) J. Appl. Bacteriol. 23, 130-135] medium (49.6 AU/ml), MPW (9.5 AU/ml) and whey (22.5 AU/ml) [1 AU/ml is the amount of antibacterial compound needed to obtain 50% growth inhibition (LD50) compared with control tubes]. In the three fed-batch cultures a shift from homolactic to mixed-acid fermentation was observed, and other products (acetic acid, butane-2,3-diol or ethanol) in addition to lactic acid were detectable in the medium. However, their contributions to the total antibacterial activity of the post-incubates (the cell-free culture supernatant obtained at the end of the fermentation process) of L. lactis CECT 539 against Carnobacterium piscicola CECT 4020 were very low.

  12. Crystallization and preliminary X-ray crystallographic analysis of β-galactosidase from Kluyveromyces lactis

    PubMed Central

    Pereira-Rodríguez, Ángel; Fernández-Leiro, Rafael; González Siso, M. Isabel; Cerdán, M. Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

    2010-01-01

    β-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the β-­galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8 Å resolution. PMID:20208165

  13. The structure and possible function of the glycolipid from Staphylococcus lactis I3

    PubMed Central

    Brundish, D. E.; Shaw, N.; Baddiley, J.

    1967-01-01

    1. The total lipid was extracted from Staphylococcus lactis I3 with chloroform–methanol mixtures and the glycolipid component was isolated by chromatography on silicic acid. 2. Saponification yielded a non-crystalline glycoside for which the structure O-β-d-glucopyranosyl-(1→6)-O-β-d-glucopyranosyl-(1→1)-d-glycerol has been established by chemical degradations and by comparison with synthetic material. 3. The role of the glycosyl diglycerides in bacterial membranes is discussed. ImagesFig. 1. PMID:5584025

  14. Fine Tuning of the Lactate and Diacetyl Production through Promoter Engineering in Lactococcus lactis

    PubMed Central

    Guo, Tingting; Kong, Jian; Zhang, Li; Zhang, Chenchen; Hu, Shumin

    2012-01-01

    Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15±0.08 mM to 9.94±0.07 mM, and the corresponding diacetyl production increased from 1.07±0.03 mM to 4.16±0.06 mM with the intracellular NADH/NAD+ ratios varying from 0.711±0.005 to 0.383±0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H2O-forming NADH oxidase activity led to 76.95% lower H2O2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H2O2 accumulation and prolong cell survival. PMID:22558426

  15. Prebiotic Effects of Agave salmiana Fructans in Lactobacillus acidophilus and Bifidobacterium lactis Cultures.

    PubMed

    Castro-Zavala, Adriana; Juárez-Flores, Bertha I; Pinos-Rodríguez, Juan M; Delgado-Portales, Rosa E; Aguirre-Rivera, Juan R; Alcocer-Gouyonnet, Francisco

    2015-11-01

    Agave salmiana is a fructan rich species that is widely distributed in Mexico. The aim of this investigation was to extract the fructans of A. salmiana and evaluate their prebiotic effect in 48 hours in vitro cultures of Bifidobacterium lactis and Lactobacillus acidophilus and to compare this effect with other available fructan sources. A significant difference in pH, optical density and biomass was found in the cultures depending on the source of fructans and the type of bacteria. It was possible to determine a dose-response effect of the A. salmiana fructans and the growth of the studied strains. PMID:26749843

  16. Prebiotic Effects of Agave salmiana Fructans in Lactobacillus acidophilus and Bifidobacterium lactis Cultures.

    PubMed

    Castro-Zavala, Adriana; Juárez-Flores, Bertha I; Pinos-Rodríguez, Juan M; Delgado-Portales, Rosa E; Aguirre-Rivera, Juan R; Alcocer-Gouyonnet, Francisco

    2015-11-01

    Agave salmiana is a fructan rich species that is widely distributed in Mexico. The aim of this investigation was to extract the fructans of A. salmiana and evaluate their prebiotic effect in 48 hours in vitro cultures of Bifidobacterium lactis and Lactobacillus acidophilus and to compare this effect with other available fructan sources. A significant difference in pH, optical density and biomass was found in the cultures depending on the source of fructans and the type of bacteria. It was possible to determine a dose-response effect of the A. salmiana fructans and the growth of the studied strains.

  17. Anti-inflammatory properties of fermented soy milk with Lactococcus lactis subsp. lactis S-SU2 in murine macrophage RAW264.7 cells and DSS-induced IBD model mice.

    PubMed

    Kawahara, Miho; Nemoto, Maki; Nakata, Toru; Kondo, Saya; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

    2015-06-01

    Six lactic acid bacteria strains (four Lactobacillus plantarum strains and one each of Lactococcus lactis subsp. lactis and Pediococcus pentosaceus) have been isolated and shown to possess anti-oxidant activity. In this study, we determined their acid, bile, salt resistance, and adhesion activity on human enterocyte-like HT-29-Luc and Caco-2 cells. An isolate Lc. lactis S-SU2 showed highest bile resistance and adhesion activity compared to type strains. S-SU2 could ferment both 10% skimmed milk and soy milk while the type strain could not ferment soy milk. Soy milk fermented with S-SU2 showed an increased nitric oxide (NO) secretion in the mouse macrophage RAW264.7 cells without bacterial lipopolysaccharide (LPS). Furthermore, the inhibitory effects of the fermented soy milk on Escherichia coli O111 LPS-induced NO secretion were higher than those of fresh soy milk. Inflammatory bowel disease (IBD) was induced in mice fed either 5% (w/v) dextran sodium sulfate (DSS) in drinking water or 50% soy milk in drinking water. Shortening of colon length, breaking of epithelial cells, lowering liver and thymus weights, and enlargement of spleen are some of the characteristics observed in the IBD, which were prevented by the use of soy milk fermented with Lc. lactis S-SU2.

  18. Anti-inflammatory properties of fermented soy milk with Lactococcus lactis subsp. lactis S-SU2 in murine macrophage RAW264.7 cells and DSS-induced IBD model mice.

    PubMed

    Kawahara, Miho; Nemoto, Maki; Nakata, Toru; Kondo, Saya; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

    2015-06-01

    Six lactic acid bacteria strains (four Lactobacillus plantarum strains and one each of Lactococcus lactis subsp. lactis and Pediococcus pentosaceus) have been isolated and shown to possess anti-oxidant activity. In this study, we determined their acid, bile, salt resistance, and adhesion activity on human enterocyte-like HT-29-Luc and Caco-2 cells. An isolate Lc. lactis S-SU2 showed highest bile resistance and adhesion activity compared to type strains. S-SU2 could ferment both 10% skimmed milk and soy milk while the type strain could not ferment soy milk. Soy milk fermented with S-SU2 showed an increased nitric oxide (NO) secretion in the mouse macrophage RAW264.7 cells without bacterial lipopolysaccharide (LPS). Furthermore, the inhibitory effects of the fermented soy milk on Escherichia coli O111 LPS-induced NO secretion were higher than those of fresh soy milk. Inflammatory bowel disease (IBD) was induced in mice fed either 5% (w/v) dextran sodium sulfate (DSS) in drinking water or 50% soy milk in drinking water. Shortening of colon length, breaking of epithelial cells, lowering liver and thymus weights, and enlargement of spleen are some of the characteristics observed in the IBD, which were prevented by the use of soy milk fermented with Lc. lactis S-SU2. PMID:25887264

  19. Occurrence of a novel L-2,4-diaminobutyrate decarboxylase activity in some species of Enterobacteriaceae, and purification and characterization of the enzymes of Enterobacter aerogenes and Serratia marcescens.

    PubMed

    Yamamoto, S; Mutoh, N; Ikai, H; Nagasaka, M

    1996-10-01

    L-2,4-Diaminobutyrate decarboxylase (DABA DC) is a novel enzyme yielding 1,3-diaminopropane (DAP) from DABA, which has previously been purified from strains of the genera Vibrio and Acinetobacter. In this study, we also detected DABA DC activity in the species of Enterobacteriaceae: E. aerogenes, E. cloacae, E. agglomerans, Serratia marcescens, S. liquefaciens, Klebsiella pneumoniace, K. oxytoca and Citrobacter freundii, all of which produced DAP in sufficient amounts. Subsequently, the DABA DCs of E. aerogenes and S. marcescens were purified to homogeneity and characterized. Two separate enzymes had similar properties with respect to chromatographic behaviors, and were a dimer with subunits of identical molecular mass of about 51 kDa. The maximal activity of each enzyme was obtained at pH 8.0-8.25. Both enzymes required pyridoxal 5'-phosphate and Mg2+ for full activity, and were highly specific for L-DABA. There was immunological similarity, but not identity between these proteins, as determined by Ouchterlony double diffusion analysis with antiserum against the E. aerogenes DABA DC. They showed the same N-terminal amino acid sequence up to the 8th residue (S-K-L-N-P-I-L-A-). These enzymes were different in molecular mass, N-terminal amino acid sequence and antigenicity from DABA DCs of Acientobacter and Vibrio species.

  20. Chimeras of mature pediocin PA-1 fused to the signal peptide of enterocin P permits the cloning, production, and expression of pediocin PA-1 in Lactococcus lactis.

    PubMed

    Martín, María; Gutiérrez, Jorge; Criado, Raquel; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2007-12-01

    Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SP(entP)) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SP(entP):pedA) and pMPP14i (SP(entP):pedA + pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti-PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SP(EntP) is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.

  1. Time-resolved genetic responses of Lactococcus lactis to a dairy environment.

    PubMed

    Bachmann, Herwig; de Wilt, Leonie; Kleerebezem, Michiel; van Hylckama Vlieg, Johan E T

    2010-05-01

    Lactococcus lactis is one of main bacterial species found in mixed dairy starter cultures for the production of semi-hard cheese. Despite the appreciation that mixed cultures are essential for the eventual properties of the manufactured cheese the vast majority of studies on L. lactis were carried out in laboratory media with a pure culture. In this study we applied an advanced recombinant in vivo expression technology (R-IVET) assay in combination with a high-throughput cheese-manufacturing protocol for the identification and subsequent validation of promoter sequences specifically induced during the manufacturing and ripening of cheese. The system allowed gene expression measurements in an undisturbed product environment without the use of antibiotics and in combination with a mixed strain starter culture. The utilization of bacterial luciferase as reporter enabled the real-time monitoring of gene expression in cheese for up to 200 h after the cheese-manufacturing process was initiated. The results revealed a number of genes that were clearly induced in cheese such as cysD, bcaP, dppA, hisC, gltA, rpsE, purL, amtB as well as a number of hypothetical genes, pseudogenes and notably genetic elements located on the non-coding strand of annotated open reading frames. Furthermore genes that are likely to be involved in interactions with bacteria used in the mixed strain starter culture were identified.

  2. Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter.

    PubMed

    Saravanan, Chinnashanmugam; Shetty, Prathap Kumar H

    2016-09-01

    Diverse exopolysaccharide (EPS)-producing isolates were isolated from an Indian acidic fermented food (Idli) based on the colony morphology. One of the EPS-producing microflora (Leuconostoc lactis KC117496) was selected for further characterization using FT-IR, HPTLC, AFM, SEM, TGA and XRD analysis. FT-IR spectroscopy revealed the α-d-glucose nature of the EPS. HPTLC analysis confirmed the presence of only glucose monomers, indicating the glucan nature of EPS. NMR spectra revealed the presence of 95% α-(1→6) and 5% branching α-(1→3) linkages. The SEM and AFM showed smooth surfaces and compact structure. TGA results showed higher degradation temperature of 272.01°C. XRD analysis proved the 33.4% crystalline nature of the EPS. Water solubility index and water-holding capacity of EPS are 14.2±0.208% and 117±7.5%. All the above characteristics of the EPS produced by L. lactis showed that the EPS is of a good-quality polysaccharide with potential applications in the food industry.

  3. The Carbohydrate Metabolism Signature of Lactococcus lactis Strain A12 Reveals Its Sourdough Ecosystem Origin

    PubMed Central

    Passerini, Delphine; Coddeville, Michèle; Le Bourgeois, Pascal; Loubière, Pascal; Ritzenthaler, Paul; Fontagné-Faucher, Catherine; Cocaign-Bousquet, Muriel

    2013-01-01

    Lactococcus lactis subsp. lactis strain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and α-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci. PMID:23872564

  4. Oligomerized backbone pilin helps piliated Lactococcus lactis to withstand shear flow.

    PubMed

    Castelain, Mickaël; Duviau, Marie-Pierre; Oxaran, Virginie; Schmitz, Philippe; Cocaign-Bousquet, Muriel; Loubière, Pascal; Piard, Jean-Christophe; Mercier-Bonin, Muriel

    2016-09-01

    The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment. PMID:27472256

  5. Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate.

    PubMed

    Chen, Jun; Shen, Jing; Ingvar Hellgren, Lars; Ruhdal Jensen, Peter; Solem, Christian

    2015-09-21

    Lactococcus lactis is essential for most cheese making, and this mesophilic bacterium has its growth optimum around 30 °C. We have, through adaptive evolution, isolated a mutant TM29 that grows well up to 39 °C, and continuous growth at 40 °C is possible if pre-incubated at a slightly lower temperature. At the maximal permissive temperature for the wild-type, 38 °C, TM29 grows 33% faster and has a 12% higher specific lactate production rate than its parent MG1363, which results in fast lactate accumulation. Genome sequencing was used to reveal the mutations accumulated, most of which were shown to affect thermal tolerance. Of the mutations with more pronounced effects, two affected expression of single proteins (chaperone; riboflavin transporter), two had pleiotropic effects (RNA polymerase) which changed the gene expression profile, and one resulted in a change in the coding sequence of CDP-diglyceride synthase. A large deletion containing 10 genes was also found to affect thermal tolerance significantly. With this study we demonstrate a simple approach to obtain non-GMO derivatives of the important L. lactis that possess properties desirable by the industry, e.g. thermal robustness and increased rate of acidification. The mutations we have identified provide a genetic basis for further investigation of thermal tolerance.

  6. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

    PubMed

    Skory, Christopher D; Côté, Gregory L

    2015-12-01

    We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

  7. Modeling of the Competitive Growth of Listeria monocytogenes and Lactococcus lactis in Vegetable Broth

    PubMed Central

    Breidt, Frederick; Fleming, Henry P.

    1998-01-01

    Current mathematical models used by food microbiologists do not address the issue of competitive growth in mixed cultures of bacteria. We developed a mathematical model which consists of a system of nonlinear differential equations describing the growth of competing bacterial cell cultures. In this model, bacterial cell growth is limited by the accumulation of protonated lactic acid and decreasing pH. In our experimental system, pure and mixed cultures of Lactococcus lactis and Listeria monocytogenes were grown in a vegetable broth medium. Predictions of the model indicate that pH is the primary factor that limits the growth of L. monocytogenes in competition with a strain of L. lactis which does not produce the bacteriocin nisin. The model also predicts the values of parameters that affect the growth and death of the competing populations. Further development of this model will incorporate the effects of additional inhibitors, such as bacteriocins, and may aid in the selection of lactic acid bacterium cultures for use in competitive inhibition of pathogens in minimally processed foods. PMID:9726854

  8. Two mitochondrial alcohol dehydrogenase activities of Kluyveromyces lactis are differently expressed during respiration and fermentation.

    PubMed

    Saliola, M; Falcone, C

    1995-12-20

    The lactose-utilizing yeast Kluyveromyces lactis is an essentially aerobic organism in which both respiration and fermentation can coexist depending on the sugar concentration. Despite a low fermentative capacity as compared to Saccharomyces cerevisiae, four structural genes encoding alcohol dehydrogenase (ADH) activities are present in this yeast. Two of these activities, namely K1ADH III and K1ADH IV, are located within mitochondria and their presence is dependent on the carbon sources in the medium. In this paper we demonstrate by transcription and activity analysis that KlADH3 is expressed in the presence of low glucose concentrations and in the presence of respiratory carbon sources other than ethanol. Indeed ethanol acts as a strong repressor of this gene. On the other hand, KlADH4 is induced by the presence of ethanol and not by other respiratory carbon sources. We also demonstrate that the presence of KLADH III and KLADH IV in K. lactis cells is dependent on glucose concentration, glucose uptake and the amount of ethanol produced. As a consequence, these activities can be used as markers for the onset of respiratory and fermentative metabolism in this yeast.

  9. Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate.

    PubMed

    Chen, Jun; Shen, Jing; Ingvar Hellgren, Lars; Ruhdal Jensen, Peter; Solem, Christian

    2015-01-01

    Lactococcus lactis is essential for most cheese making, and this mesophilic bacterium has its growth optimum around 30 °C. We have, through adaptive evolution, isolated a mutant TM29 that grows well up to 39 °C, and continuous growth at 40 °C is possible if pre-incubated at a slightly lower temperature. At the maximal permissive temperature for the wild-type, 38 °C, TM29 grows 33% faster and has a 12% higher specific lactate production rate than its parent MG1363, which results in fast lactate accumulation. Genome sequencing was used to reveal the mutations accumulated, most of which were shown to affect thermal tolerance. Of the mutations with more pronounced effects, two affected expression of single proteins (chaperone; riboflavin transporter), two had pleiotropic effects (RNA polymerase) which changed the gene expression profile, and one resulted in a change in the coding sequence of CDP-diglyceride synthase. A large deletion containing 10 genes was also found to affect thermal tolerance significantly. With this study we demonstrate a simple approach to obtain non-GMO derivatives of the important L. lactis that possess properties desirable by the industry, e.g. thermal robustness and increased rate of acidification. The mutations we have identified provide a genetic basis for further investigation of thermal tolerance. PMID:26388459

  10. Effectiveness of thermal treatments and biocides in the inactivation of Argentinian Lactococcus lactis phages.

    PubMed

    Suárez, Viviana B; Reinheimer, Jorge A

    2002-11-01

    The thermal and chemical resistance levels of four autochthonal bacteriophages of Lactococcus lactis subsp. lactis, isolated from cheese processes, was investigated. The times required to obtain 99% inactivation of phages (T99) at 63 and 72 degrees C in three suspension media (M17 broth, reconstituted commercial nonfat skim milk, and Tris magnesium gelatin buffer) were determined. Thermal resistance was dependent on the phage studied, and the results of this study demonstrate that pasteurization treatments used in dairy industries may leave viable viral particles in milk. It was possible to determine that M17 broth was generally the least protective medium, while phosphate buffer was the most protective one. Peracetic acid (0.15%, vol/vol) was the most effective viricidal agent, with exposures of 5 min being sufficient to inactivate high-titer phage suspensions (>10(6) PFU/ml). To achieve total inactivation (<10 PFU/ml) of viral suspensions, sodium hypochlorite was effective at 100 ppm for only two phages, while the other two phages needed concentrations of 200 and 300 ppm. Ethanol at concentrations of 100 and 75% proved to be very efficient in inactivating phages, but isopropanol was not effective against them.

  11. Statistical investigation of Kluyveromyces lactis cells permeabilization with ethanol by response surface methodology

    PubMed Central

    de Faria, Janaína T.; Rocha, Pollyana F.; Converti, Attilio; Passos, Flávia M.L.; Minim, Luis A.; Sampaio, Fábio C.

    2013-01-01

    The aim of our study was to select the optimal operating conditions to permeabilize Kluyveromyces lactis cells using ethanol as a solvent as an alternative to cell disruption and extraction. Cell permeabilization was carried out by a non-mechanical method consisting of chemical treatment with ethanol, and the results were expressed as β-galactosidase activity. Experiments were conducted under different conditions of ethanol concentration, treatment time and temperature according to a central composite rotatable design (CCRD), and the collected results were then worked out by response surface methodology (RSM). Cell permeabilization was improved by an increase in ethanol concentration and simultaneous decreases in the incubation temperature and treatment time. Such an approach allowed us to identify an optimal range of the independent variables within which the β-galactosidase activity was optimized. A maximum permeabilization of 2,816 mmol L−1 oNP min−1 g−1 was obtained by treating cells with 75.0% v/v of ethanol at 20.0 °C for 15.0 min. The proposed methodology resulted to be effective and suited for K. lactis cells permeabilization at a lab-scale and promises to be of possible interest for future applications mainly in the food industry. PMID:24688494

  12. Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate

    PubMed Central

    Chen, Jun; Shen, Jing; Ingvar Hellgren, Lars; Ruhdal Jensen, Peter; Solem, Christian

    2015-01-01

    Lactococcus lactis is essential for most cheese making, and this mesophilic bacterium has its growth optimum around 30 °C. We have, through adaptive evolution, isolated a mutant TM29 that grows well up to 39 °C, and continuous growth at 40 °C is possible if pre-incubated at a slightly lower temperature. At the maximal permissive temperature for the wild-type, 38 °C, TM29 grows 33% faster and has a 12% higher specific lactate production rate than its parent MG1363, which results in fast lactate accumulation. Genome sequencing was used to reveal the mutations accumulated, most of which were shown to affect thermal tolerance. Of the mutations with more pronounced effects, two affected expression of single proteins (chaperone; riboflavin transporter), two had pleiotropic effects (RNA polymerase) which changed the gene expression profile, and one resulted in a change in the coding sequence of CDP-diglyceride synthase. A large deletion containing 10 genes was also found to affect thermal tolerance significantly. With this study we demonstrate a simple approach to obtain non-GMO derivatives of the important L. lactis that possess properties desirable by the industry, e.g. thermal robustness and increased rate of acidification. The mutations we have identified provide a genetic basis for further investigation of thermal tolerance. PMID:26388459

  13. Isolation and characterization of exopolysaccharide from Leuconostoc lactis KC117496 isolated from idli batter.

    PubMed

    Saravanan, Chinnashanmugam; Shetty, Prathap Kumar H

    2016-09-01

    Diverse exopolysaccharide (EPS)-producing isolates were isolated from an Indian acidic fermented food (Idli) based on the colony morphology. One of the EPS-producing microflora (Leuconostoc lactis KC117496) was selected for further characterization using FT-IR, HPTLC, AFM, SEM, TGA and XRD analysis. FT-IR spectroscopy revealed the α-d-glucose nature of the EPS. HPTLC analysis confirmed the presence of only glucose monomers, indicating the glucan nature of EPS. NMR spectra revealed the presence of 95% α-(1→6) and 5% branching α-(1→3) linkages. The SEM and AFM showed smooth surfaces and compact structure. TGA results showed higher degradation temperature of 272.01°C. XRD analysis proved the 33.4% crystalline nature of the EPS. Water solubility index and water-holding capacity of EPS are 14.2±0.208% and 117±7.5%. All the above characteristics of the EPS produced by L. lactis showed that the EPS is of a good-quality polysaccharide with potential applications in the food industry. PMID:25687478

  14. Ingestion of milk fermented by genetically modified Lactococcus lactis improves the riboflavin status of deficient rats.

    PubMed

    LeBlanc, J G; Burgess, C; Sesma, F; Savoy de Giori, G; van Sinderen, D

    2005-10-01

    Riboflavin deficiency is common in many parts of the world, particularly in developing countries. The use of riboflavin-producing strains in the production of dairy products such as fermented milks, yogurts, and cheeses is feasible and economically attractive because it would decrease the costs involved during conventional vitamin fortification and satisfy consumer demands for healthier foods. The present study was conducted to assess in a rat bioassay the response of administration of milk fermented by modified Lactococcus lactis on the riboflavin status of deficient rats. Rats were fed a riboflavin-deficient diet during 21 d after which this same diet was supplemented with milk fermented by Lactoccus lactis pNZGBAH, a strain that overproduces riboflavin during fermentation. The novel fermented product, with increased levels of riboflavin, was able to eliminate most physiological manifestations of ariboflavinosis, such as stunted growth, elevated erythrocyte glutathione reductase activation coefficient values and hepatomegaly, that were observed using a riboflavin depletion-repletion model, whereas a product fermented with a nonriboflavin-producing strain did not show similar results. A safety assessment of this modified strain was performed by feeding rodents with the modified strain daily for 4 wk. This strain caused no detectable secondary effects. These results pave the way for analyzing the effect of similar riboflavin-overproducing lactic acid bacteria in human trials. The regular consumption of products with increased levels of riboflavin could help prevent deficiencies of this essential vitamin.

  15. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

    PubMed

    Skory, Christopher D; Côté, Gregory L

    2015-12-01

    We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme. PMID:26239071

  16. Transcription profiling of interactions between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 during Cheddar cheese simulation.

    PubMed

    Desfossés-Foucault, Émilie; LaPointe, Gisèle; Roy, Denis

    2014-05-16

    The starter cultures (Lactococcus sp.) and non-starter lactic acid bacteria (mostly Lactobacillus spp.) are essential to flavor development of Cheddar cheese. The aim of this study was to elucidate the transcriptional interaction between Lactococcus lactis subsp. cremoris SK11 and Lactobacillus paracasei ATCC 334 in mixed cultures during simulated Cheddar cheese manufacture (Pearce activity test) and ripening (slurry). Reverse transcription quantitative PCR (RT-qPCR) was used to quantify the expression of 34 genes common to both bacteria and for eight genes specific to either L. lactis subsp. cremoris SK11 or L. paracasei ATCC 334. The multifactorial analysis (MFA) performed on fold change results for each gene revealed that the genes linked to stress, protein and peptide degradation as well as carbohydrate metabolism of L. paracasei ATCC 334 were especially overexpressed in mixed culture with L. lactis subsp. cremoris SK11 during the ripening simulation. For L. lactis subsp. cremoris SK11, genes coding for amino acid metabolism were more expressed during the cheese manufacture simulation, especially in single culture. These results show how complementary functions of starter and NSLAB contribute to activities useful for flavor development. PMID:24674930

  17. Oral immunization with live Lactococcus lactis expressing rotavirus VP8 subunit induces specific immune response in mice.

    PubMed

    Marelli, Belkis; Perez, Ana Rosa; Banchio, Claudia; de Mendoza, Diego; Magni, Christian

    2011-07-01

    Rotaviruses are the major cause of worldwide infectious diarrhea in children and vaccination is considered to be the most effective way to control these infections. The development of a mucosal live vaccine using the food-grade lactic acid bacteria Lactococcus lactis as antigen vehicle is an attractive and safe vaccination strategy against rotavirus. In this study, the construction of recombinant L. lactis strains able to produce the rotavirus spike-protein subunit VP8 in cytoplasmic, secreted and cell wall-anchored forms is reported. Evaluation of the immune response generated after immunization was conducted in a mouse model. The present study shows that animals inoculated orally with the L. lactis strain producing the cytoplasmic form of VP8 (LL1) developed significant levels of intestinal IgA antibodies while animals receiving L. lactis producing the cell wall-anchored VP8 form (LL3) exhibited anti-VP8 antibodies at both intestinal and systemic levels. Furthermore, it was observed that intestinal antibodies of the LL1-treated group and serum antibodies of the LL3-treated group were able to block rotavirus infection by 50% and 100%, respectively. These encouraging results represent a step towards the development of a new and safe mucosal vaccine against rotavirus.

  18. Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm

    PubMed Central

    de Oliveira, Viviane Matoso; de Almeida Pina, André Vicioli; Pérez-Chaparro, Paula Juliana; de Almeida, Lara Mendes; de Vasconcelos, Janaina Mota; de Oliveira, Layanna Freitas; da Silva, Daisy Elaine Andrade; Rogez, Hervé Louis Ghislain; Cretenet, Marina; Mamizuka, Elsa Masae; Nunes, Marcio Roberto Teixeira

    2014-01-01

    We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product. PMID:25414513

  19. Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm.

    PubMed

    McCulloch, John Anthony; de Oliveira, Viviane Matoso; de Almeida Pina, André Vicioli; Pérez-Chaparro, Paula Juliana; de Almeida, Lara Mendes; de Vasconcelos, Janaina Mota; de Oliveira, Layanna Freitas; da Silva, Daisy Elaine Andrade; Rogez, Hervé Louis Ghislain; Cretenet, Marina; Mamizuka, Elsa Masae; Nunes, Marcio Roberto Teixeira

    2014-11-20

    We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product.

  20. Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system.

    PubMed

    Kördikanlıoğlu, Burcu; Şimşek, Ömer; Saris, Per E J

    2015-01-01

    In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed-batch fermentation system with high fidelity (R(2) 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L(-1) h(-1) , 3 μg mL(-1) and 40%, respectively. While 1711 IU mL(-1) nisin was produced by L. lactis N8 in control fed-batch fermentation, 5410 IU mL(-1) nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed-batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed-batch fermentation. PMID:25826783

  1. Genotypic and Phenotypic Analysis of Dairy Lactococcus lactis Biodiversity in Milk: Volatile Organic Compounds as Discriminating Markers

    PubMed Central

    Dhaisne, Amandine; Guellerin, Maeva; Laroute, Valérie; Laguerre, Sandrine; Le Bourgeois, Pascal; Loubiere, Pascal

    2013-01-01

    The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic diversity, 82 variables were selected as important dairy features; they included physiological descriptors and the production of metabolites and volatile organic compounds (VOCs). Principal-component analysis (PCA) demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination. A method of variable selection was developed to reduce the time-consuming experimentation. We therefore identified 20 variables, all associated with VOCs, as phenotypic markers allowing discrimination between strain groups. These markers are representative of the three metabolic pathways involved in flavor: lipolysis, proteolysis, and glycolysis. Despite great phenotypic diversity, the strains could be divided into four robust phenotypic clusters based on their metabolic orientations. Inclusion of genotypic diversity in addition to phenotypic characters in the classification led to five clusters rather than four being defined. However, genotypic characters make a smaller contribution than phenotypic variables (no genetic distances selected among the most contributory variables). This work proposes an original method for the phenotypic differentiation of closely related strains in milk and may be the first step toward a predictive classification for the manufacture of starters. PMID:23709512

  2. Effect of autochthonous bacteriocin-producing Lactococcus lactis on bacterial population dynamics and growth of halotolerant bacteria in Brazilian charqui.

    PubMed

    Biscola, Vanessa; Abriouel, Hikmate; Todorov, Svetoslav Dimitrov; Capuano, Verena Sant'Anna Cabral; Gálvez, Antonio; Franco, Bernadette Dora Gombossy de Melo

    2014-12-01

    Charqui is a fermented, salted and sun-dried meat product, widely consumed in Brazil and exported to several countries. Growth of microorganisms in this product is unlikely due to reduced Aw, but halophilic and halotolerant bacteria may grow and cause spoilage. Charqui is a good source of lactic acid bacteria able to produce antimicrobial bacteriocins. In this study, an autochthonous bacteriocinogenic strain (Lactococcus lactis subsp. lactis 69), isolated from charqui, was added to the meat used for charqui manufacture and evaluated for its capability to prevent the growth of spoilage bacteria during storage up to 45 days. The influence of L. lactis 69 on the bacterial diversity during the manufacturing of the product was also studied, using denaturing gradient gel electrophoresis (DGGE). L. lactis 69 did not affect the counts and diversity of lactic acid bacteria during manufacturing and storage, but influenced negatively the populations of halotolerant microorganisms, reducing the spoilage potential. The majority of tested virulence genes was absent, evidencing the safety and potential technological application of this strain as an additional hurdle to inhibit undesirable microbial growth in this and similar fermented meat products.

  3. An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

    PubMed Central

    Romagnoli, G; Verhoeven, M D; Mans, R; Fleury Rey, Y; Bel-Rhlid, R; van den Broek, M; Maleki Seifar, R; Ten Pierick, A; Thompson, M; Müller, V; Wahl, S A; Pronk, J T; Daran, J M

    2014-01-01

    Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. PMID:24912400

  4. Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.

    PubMed

    Rezende, Rafael M; Oliveira, Rafael P; Medeiros, Samara R; Gomes-Santos, Ana C; Alves, Andrea C; Loli, Flávia G; Guimarães, Mauro A F; Amaral, Sylvia S; da Cunha, André P; Weiner, Howard L; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M C

    2013-02-01

    Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

  5. Chemically defined media and auxotrophy of the prolific l-lactic acid producer Lactococcus lactis IO-1.

    PubMed

    Machii, Miki; Watanabe, Satoru; Zendo, Takeshi; Chibazakura, Taku; Sonomoto, Kenji; Shimizu-Kadota, Mariko; Yoshikawa, Hirofumi

    2013-05-01

    Two chemically defined media, CDM-1G and CDM-1X, that use glucose and xylose as carbon sources, respectively, were prepared for Lactococcus lactis strain IO-1. The maximal cell density at 600 nm in CDM-1G exceeded 2. Omission growth experiments indicated that IO-1 is auxotrophic for 2 vitamins and 6 amino acids.

  6. Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

    PubMed Central

    Rezende, Rafael M.; Oliveira, Rafael P.; Medeiros, Samara R.; Gomes-Santos, Ana C.; Alves, Andrea C.; Loli, Flávia G.; Guimarães, Mauro A.F.; Amaral, Sylvia S.; da Cunha, André P.; Weiner, Howard L.; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M.C.

    2013-01-01

    Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

  7. Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.

    PubMed

    Rezende, Rafael M; Oliveira, Rafael P; Medeiros, Samara R; Gomes-Santos, Ana C; Alves, Andrea C; Loli, Flávia G; Guimarães, Mauro A F; Amaral, Sylvia S; da Cunha, André P; Weiner, Howard L; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M C

    2013-02-01

    Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice.

  8. Interaction of Bifidobacterium animalis subspecies lactis (Bb 12) and Salmonella typhimurium in continuous-flow chemostatic culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A commercially available probiotic, Bifidobacterium animalis subspecies lactis (Bb12) was adapted to and maintained in a continuous-flow chemostat culture. We evaluated the growth characteristics and interactive effects of Bb12 and a porcine-derived Salmonella typhimurium (St) when cultivated singly...

  9. Interaction of Bifidobacterium animalis subspecies lactis (Bb12) and Salmonella typhimurium in continuous-flow chemostatic culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A commercially available probiotic, Bifidobacterium animalis subspecies lactis (Bb12) was adapted to and maintained in a continuous-flow chemostat culture. We evaluated the growth characteristics and in interactive effects of Bb12 and a porcine-derived Salmonella typhimurium (St) when cultivated si...

  10. Nisin production of Lactococcus lactis N8 with hemin-stimulated cell respiration in fed-batch fermentation system.

    PubMed

    Kördikanlıoğlu, Burcu; Şimşek, Ömer; Saris, Per E J

    2015-01-01

    In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed-batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed-batch fermentation system with high fidelity (R(2) 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L(-1) h(-1) , 3 μg mL(-1) and 40%, respectively. While 1711 IU mL(-1) nisin was produced by L. lactis N8 in control fed-batch fermentation, 5410 IU mL(-1) nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed-batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed-batch fermentation.

  11. Essential oils affect populations of some rumen bacteria in vitro as revealed by microarray (RumenBactArray) analysis.

    PubMed

    Patra, Amlan K; Yu, Zhongtang

    2015-01-01

    In a previous study origanum oil (ORO), garlic oil (GAO), and peppermint oil (PEO) were shown to effectively lower methane production, decrease abundance of methanogens, and change abundances of several bacterial populations important to feed digestion in vitro. In this study, the impact of these essential oils (EOs, at 0.50 g/L) on the rumen bacterial community composition and population was further examined using the recently developed RumenBactArray. Species richness (expressed as number of operational taxonomic units, OTUs) in the phylum Firmicutes, especially those in the class Clostridia, was decreased by ORO and GAO, but increased by PEO, while that in the phylum Bacteroidetes was increased by ORO and PEO. Species richness in the genus Butyrivibrio was lowered by all the EOs. Increases of Bacteroidetes OTUs mainly resulted from increases of Prevotella OTUs. Overall, 67 individual OTUs showed significant differences (P ≤ 0.05) in relative abundance across the EO treatments. The predominant OTUs affected by EOs were diverse, including those related to Syntrophococcus sucromutans, Succiniclasticum ruminis, and Lachnobacterium bovis, and those classified to Prevotella, Clostridium, Roseburia, Pseudobutyrivibrio, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Bacteroidales, and Clostridiales. In total, 60 OTUs were found significantly (P ≤ 0.05) correlated with feed degradability, ammonia concentration, and molar percentage of volatile fatty acids. Taken together, this study demonstrated extensive impact of EOs on rumen bacterial communities in an EO type-dependent manner, especially those in the predominant families Prevotellaceae, Lachnospiraceae, and Ruminococcaceae. The information from this study may aid in understanding the effect of EOs on feed digestion and fermentation by rumen bacteria. PMID:25914694

  12. Essential oils affect populations of some rumen bacteria in vitro as revealed by microarray (RumenBactArray) analysis

    PubMed Central

    Patra, Amlan K.; Yu, Zhongtang

    2015-01-01

    In a previous study origanum oil (ORO), garlic oil (GAO), and peppermint oil (PEO) were shown to effectively lower methane production, decrease abundance of methanogens, and change abundances of several bacterial populations important to feed digestion in vitro. In this study, the impact of these essential oils (EOs, at 0.50 g/L) on the rumen bacterial community composition and population was further examined using the recently developed RumenBactArray. Species richness (expressed as number of operational taxonomic units, OTUs) in the phylum Firmicutes, especially those in the class Clostridia, was decreased by ORO and GAO, but increased by PEO, while that in the phylum Bacteroidetes was increased by ORO and PEO. Species richness in the genus Butyrivibrio was lowered by all the EOs. Increases of Bacteroidetes OTUs mainly resulted from increases of Prevotella OTUs. Overall, 67 individual OTUs showed significant differences (P ≤ 0.05) in relative abundance across the EO treatments. The predominant OTUs affected by EOs were diverse, including those related to Syntrophococcus sucromutans, Succiniclasticum ruminis, and Lachnobacterium bovis, and those classified to Prevotella, Clostridium, Roseburia, Pseudobutyrivibrio, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Bacteroidales, and Clostridiales. In total, 60 OTUs were found significantly (P ≤ 0.05) correlated with feed degradability, ammonia concentration, and molar percentage of volatile fatty acids. Taken together, this study demonstrated extensive impact of EOs on rumen bacterial communities in an EO type-dependent manner, especially those in the predominant families Prevotellaceae, Lachnospiraceae, and Ruminococcaceae. The information from this study may aid in understanding the effect of EOs on feed digestion and fermentation by rumen bacteria. PMID:25914694

  13. Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons.

    PubMed

    Ballal, Sonia A; Veiga, Patrick; Fenn, Kathrin; Michaud, Monia; Kim, Jason H; Gallini, Carey Ann; Glickman, Jonathan N; Quéré, Gaëlle; Garault, Peggy; Béal, Chloé; Derrien, Muriel; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre; van Hylckama Vlieg, Johan; Garrett, Wendy S

    2015-06-23

    Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet(-/-) Rag2(-/-) mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique to L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen results in reactive oxygen species (ROS) generation. Also, ROS are produced at high levels during intestinal inflammation and cause tissue damage. L. lactis I-1631 possesses genes encoding enzymes that detoxify ROS, such as superoxide dismutase (SodA). Thus, we hypothesized that lactococcal SodA played a role in attenuating colitis. Inactivation of the sodA gene abolished L. lactis I-1631's beneficial effect in the T-bet(-/-) Rag2(-/-) model. Similar effects were obtained in two additional colonic inflammation models, Il10(-/-) mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O2 (-)) can be detoxified by cytoplasmic lactoccocal SodA led to the finding that host antimicrobial-mediated lysis is a prerequisite for SodA release and SodA's extracytoplasmic O2 (-) scavenging. L. lactis I-1631 may represent a promising vehicle to deliver antioxidant, colitis-attenuating SodA to the inflamed intestinal mucosa, and host antimicrobials may play a critical role in mediating SodA's bioaccessibility.

  14. Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons

    PubMed Central

    Ballal, Sonia A.; Veiga, Patrick; Fenn, Kathrin; Michaud, Monia; Kim, Jason H.; Gallini, Carey Ann; Glickman, Jonathan N.; Quéré, Gaëlle; Garault, Peggy; Béal, Chloé; Derrien, Muriel; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre; van Hylckama Vlieg, Johan; Garrett, Wendy S.

    2015-01-01

    Beneficial microbes that target molecules and pathways, such as oxidative stress, which can negatively affect both host and microbiota, may hold promise as an inflammatory bowel disease therapy. Prior work showed that a five-strain fermented milk product (FMP) improved colitis in T-bet−/− Rag2−/− mice. By varying the number of strains used in the FMP, we found that Lactococcus lactis I-1631 was sufficient to ameliorate colitis. Using comparative genomic analyses, we identified genes unique to L. lactis I-1631 involved in oxygen respiration. Respiration of oxygen results in reactive oxygen species (ROS) generation. Also, ROS are produced at high levels during intestinal inflammation and cause tissue damage. L. lactis I-1631 possesses genes encoding enzymes that detoxify ROS, such as superoxide dismutase (SodA). Thus, we hypothesized that lactococcal SodA played a role in attenuating colitis. Inactivation of the sodA gene abolished L. lactis I-1631’s beneficial effect in the T-bet−/− Rag2−/− model. Similar effects were obtained in two additional colonic inflammation models, Il10−/− mice and dextran sulfate sodium-treated mice. Efforts to understand how a lipophobic superoxide anion (O2−) can be detoxified by cytoplasmic lactoccocal SodA led to the finding that host antimicrobial-mediated lysis is a prerequisite for SodA release and SodA’s extracytoplasmic O2− scavenging. L. lactis I-1631 may represent a promising vehicle to deliver antioxidant, colitis-attenuating SodA to the inflamed intestinal mucosa, and host antimicrobials may play a critical role in mediating SodA’s bioaccessibility. PMID:26056274

  15. Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis.

    PubMed

    Wang, Hui; Zhang, Liang; Shi, Guiyang

    2015-12-01

    The pla2 gene encoding a phospholipase A2 (EC 3.1.1.4) of Lactobacillus casei DSM20011 was cloned and expressed in the yeast Kluyveromyces lactis GG799 successfully for the first time. The structural pla2 gene fused in frame with the K. lactis secretion signal α-mating factor was integrated into the LAC4 locus and expressed under the control of the LAC4 promoter. sPLA2 activity was detected in the culture supernatant during shake flask culture of K. lactis/pKLAC1-pla2. In comparison with the control strain K. lactis/pKLAC1, SDS-PAGE analysis revealed a 17-kDa recombinant protein band in K. lactis/pKLAC1-pla2, which was consistent with the predicted molecular weight of the mature protein. Real-time quantitative PCR analysis indicated that the copy number of the integrated pla2 gene ranged from 2 to 6 and positively correlated with sPLA2 activity. When the inducer galactose was used as the carbon source, the sPLA2 activity in the culture supernatant of the recombinant that harbored six pla2 gene copies reached 1.96 ± 0.15 U/mL. The influence of the culture composition and conditions on the recombinant sPLA2 activity in shake flask culture were also studied. When the recombinant was cultured at 30°C in a YPD medium culture volume of 70 mL in a 250-mL shake flask with an initial pH of 7.0, the sPLA2 activity reached 2.16 ± 0.18 U/mL.

  16. Complete Sequences of Four Plasmids of Lactococcus lactis subsp. cremoris SK11 Reveal Extensive Adaptation to the Dairy Environment†

    PubMed Central

    Siezen, Roland J.; Renckens, Bernadet; van Swam, Iris; Peters, Sander; van Kranenburg, Richard; Kleerebezem, Michiel; de Vos, Willem M.

    2005-01-01

    Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted α-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these “adaptation” genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer. PMID:16332824

  17. Versatile vector suite for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis.

    PubMed

    Neef, Jolanda; Milder, Fin J; Koedijk, Danny G A M; Klaassens, Marindy; Heezius, Erik C; van Strijp, Jos A G; Otto, Andreas; Becher, Dörte; van Dijl, Jan Maarten; Buist, Girbe

    2015-11-01

    Recent studies have shown that the Gram-positive bacterium Lactococcus lactis can be exploited for the expression of heterologous proteins; however, a versatile set of vectors suitable for inducible extracellular protein production and subsequent purification of the expressed proteins by immobilized metal affinity chromatography was so far lacking. Here we describe three novel vectors that, respectively, facilitate the nisin-inducible production of N- or C-terminally hexa-histidine (His6)-tagged proteins in L. lactis. One of these vectors also encodes a tobacco etch virus (TEV) protease cleavage site allowing removal of the N-terminal His6-tag from expressed proteins. Successful application of the developed vectors for protein expression, purification and/or functional studies is exemplified with six different cell wall-bound or secreted proteins from Staphylococcus aureus. The results show that secretory production of S. aureus proteins is affected by the position, N- or C-terminal, of the His6-tag. This seems to be due to an influence of the His6-tag on protein stability. Intriguingly, the S. aureus IsdB protein, which is phosphorylated in S. aureus, was also found to be phosphorylated when heterologously produced in L. lactis, albeit not on the same Tyr residue. This implies that this particular post-translational protein modification is to some extent conserved in S. aureus and L. lactis. Altogether, we are confident that the present vector set combined with the L. lactis expression host has the potential to become a very useful tool in optimization of the expression, purification and functional analysis of extracytoplasmic bacterial proteins. PMID:26160391

  18. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism.

    PubMed

    van der Meulen, Sjoerd B; de Jong, Anne; Kok, Jan

    2016-01-01

    RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species. PMID:26950529

  19. Transcriptome landscape of Lactococcus lactis reveals many novel RNAs including a small regulatory RNA involved in carbon uptake and metabolism

    PubMed Central

    van der Meulen, Sjoerd B.; de Jong, Anne; Kok, Jan

    2016-01-01

    ABSTRACT RNA sequencing has revolutionized genome-wide transcriptome analyses, and the identification of non-coding regulatory RNAs in bacteria has thus increased concurrently. Here we reveal the transcriptome map of the lactic acid bacterial paradigm Lactococcus lactis MG1363 by employing differential RNA sequencing (dRNA-seq) and a combination of manual and automated transcriptome mining. This resulted in a high-resolution genome annotation of L. lactis and the identification of 60 cis-encoded antisense RNAs (asRNAs), 186 trans-encoded putative regulatory RNAs (sRNAs) and 134 novel small ORFs. Based on the putative targets of asRNAs, a novel classification is proposed. Several transcription factor DNA binding motifs were identified in the promoter sequences of (a)sRNAs, providing insight in the interplay between lactococcal regulatory RNAs and transcription factors. The presence and lengths of 14 putative sRNAs were experimentally confirmed by differential Northern hybridization, including the abundant RNA 6S that is differentially expressed depending on the available carbon source. For another sRNA, LLMGnc_147, functional analysis revealed that it is involved in carbon uptake and metabolism. L. lactis contains 13% leaderless mRNAs (lmRNAs) that, from an analysis of overrepresentation in GO classes, seem predominantly involved in nucleotide metabolism and DNA/RNA binding. Moreover, an A-rich sequence motif immediately following the start codon was uncovered, which could provide novel insight in the translation of lmRNAs. Altogether, this first experimental genome-wide assessment of the transcriptome landscape of L. lactis and subsequent sRNA studies provide an extensive basis for the investigation of regulatory RNAs in L. lactis and related lactococcal species. PMID:26950529

  20. Secretory expression of a phospholipase A2 from Lactobacillus casei DSM20011 in Kluyveromyces lactis.

    PubMed

    Wang, Hui; Zhang, Liang; Shi, Guiyang

    2015-12-01

    The pla2 gene encoding a phospholipase A2 (EC 3.1.1.4) of Lactobacillus casei DSM20011 was cloned and expressed in the yeast Kluyveromyces lactis GG799 successfully for the first time. The structural pla2 gene fused in frame with the K. lactis secretion signal α-mating factor was integrated into the LAC4 locus and expressed under the control of the LAC4 promoter. sPLA2 activity was detected in the culture supernatant during shake flask culture of K. lactis/pKLAC1-pla2. In comparison with the control strain K. lactis/pKLAC1, SDS-PAGE analysis revealed a 17-kDa recombinant protein band in K. lactis/pKLAC1-pla2, which was consistent with the predicted molecular weight of the mature protein. Real-time quantitative PCR analysis indicated that the copy number of the integrated pla2 gene ranged from 2 to 6 and positively correlated with sPLA2 activity. When the inducer galactose was used as the carbon source, the sPLA2 activity in the culture supernatant of the recombinant that harbored six pla2 gene copies reached 1.96 ± 0.15 U/mL. The influence of the culture composition and conditions on the recombinant sPLA2 activity in shake flask culture were also studied. When the recombinant was cultured at 30°C in a YPD medium culture volume of 70 mL in a 250-mL shake flask with an initial pH of 7.0, the sPLA2 activity reached 2.16 ± 0.18 U/mL. PMID:26108160

  1. Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

    2013-09-01

    Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

  2. Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe[superscript 2+] metal-ion preference

    SciTech Connect

    Jackson, Colin J.; Hadler, Kieran S.; Carr, Paul D.; Oakley, Aaron J.; Yip, Sylvia; Schenk, Gerhard; Ollis, David L.

    2011-09-28

    The structure of a malonate-bound form of the glycerophosphodiesterase from Enterobacter aerogenes, GpdQ, has been refined at a resolution of 2.2 {angstrom} to a final R factor of 17.1%. The structure was originally solved to 2.9 {angstrom} resolution using SAD phases from Zn{sup 2+} metal ions introduced into the active site of the apoenzyme [Jackson et al. (2007), J. Mol. Biol. 367, 1047-1062]. However, the 2.9 {angstrom} resolution was insufficient to discern significant details of the architecture of the binuclear metal centre that constitutes the active site. Furthermore, kinetic analysis revealed that the enzyme lost a significant amount of activity in the presence of Zn2+, suggesting that it is unlikely to be a catalytically relevant metal ion. In this communication, a higher resolution structure of GpdQ is presented in which malonate is visibly coordinated in the active site and analysis of the native metal-ion preference is presented using atomic absorption spectroscopy and anomalous scattering. Catalytic implications of the structure and its Fe{sup 2+} metal-ion preference are discussed.

  3. NDM-1 encoded by a pNDM-BJ01-like plasmid p3SP-NDM in clinical Enterobacter aerogenes.

    PubMed

    Chen, Zhenhong; Li, Hongxia; Feng, Jiao; Li, Yuxue; Chen, Xin; Guo, Xuemin; Chen, Weijun; Wang, Li; Lin, Lei; Yang, Huiying; Yang, Wenhui; Wang, Jie; Zhou, Dongsheng; Liu, Changting; Yin, Zhe

    2015-01-01

    A carbapenem-nonsusceptible Enterobacter aerogenes strain named 3-SP was isolated from a human case of pneumonia in a Chinese teaching hospital. NDM-1 carbapenemase is produced by a pNDM-BJ01-like conjugative plasmid designated p3SP-NDM to account for carbapenem resistance of 3-SP. p3SP-NDM was fully sequenced and compared with all publically available pNDM-BJ01-like plasmids. The genetic differences between p3SP-NDM and pNDM-BJ01 include only 18 single nucleotide polymorphisms, a 1 bp deletion and a 706 bp deletion. p3SP-NDM and pNDM-BJ01 harbor an identical Tn125 element organized as ISAba125, bla NDM-1, ble MBL, ΔtrpF, dsbC, cutA, ΔgroES, groEL, ISCR27, and ISAba125. The bla NDM-1 surrounding regions in these pNDM-BJ01-like plasmids have a conserved linear organization ISAba14-aphA6-Tn125-unknown IS, with considerable genetic differences identified within or immediately downstream of Tn125. All reported pNDM-BJ01-like plasmids are exclusively found in Acinetobacter, whereas this is the first report of identification of a pNDM-BJ01-like plasmid in Enterobacteriaceae.

  4. Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe[supscript 2+] metal-ion preference

    SciTech Connect

    Jackson, Colin J.; Hadler, Kieran S.; Carr, Paul D.; Oakley, Aaron J.; Yip, Sylvia; Schenk, Gerhard; Ollis, David L.

    2010-09-20

    The structure of a malonate-bound form of the glycerophosphodiesterase from Enterobacter aerogenes, GpdQ, has been refined at a resolution of 2.2 {angstrom} to a final R factor of 17.1%. The structure was originally solved to 2.9 {angstrom} resolution using SAD phases from Zn{sup 2+} metal ions introduced into the active site of the apoenzyme [Jackson et al. (2007), J. Mol. Biol. 367, 1047-1062]. However, the 2.9 {angstrom} resolution was insufficient to discern significant details of the architecture of the binuclear metal centre that constitutes the active site. Furthermore, kinetic analysis revealed that the enzyme lost a significant amount of activity in the presence of Zn{sup 2+}, suggesting that it is unlikely to be a catalytically relevant metal ion. In this communication, a higher resolution structure of GpdQ is presented in which malonate is visibly coordinated in the active site and analysis of the native metal-ion preference is presented using atomic absorption spectroscopy and anomalous scattering. Catalytic implications of the structure and its Fe{sup 2+} metal-ion preference are discussed.

  5. Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis.

    PubMed

    Li, Peng-cheng; Qiao, Xu-wen; Zheng, Qi-sheng; Hou, Ji-bo

    2016-01-27

    The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P<0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P<0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P<0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets. PMID

  6. Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

    PubMed

    Šiekštelė, Rimantas; Veteikytė, Aušra; Tvaska, Bronius; Matijošytė, Inga

    2015-10-01

    Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability. PMID:26254038

  7. Growth kinetics and physiological behavior of co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis, fermenting carob sugars extracted with whey.

    PubMed

    Rodrigues, B; Lima-Costa, M E; Constantino, A; Raposo, S; Felizardo, C; Gonçalves, D; Fernandes, T; Dionísio, L; Peinado, J M

    2016-10-01

    Alcoholic fermentation of carob waste sugars (sucrose, glucose and fructose) extracted with cheese whey, by co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis has been analyzed. Growth and fermentation of S. cerevisiae in the carob-whey medium showed an inhibition of about 30% in comparison with water-extracted carob. The inhibition of K. lactis on carob-whey was greater (70%) when compared with the whey medium alone, due to osmolarity problems. Oxygen availability was a very important factor for K. lactis, influencing its fermentation performance. When K. lactis was grown alone on carob-whey medium, lactose was always consumed first, and glucose and fructose were consumed afterwards, only at high aeration conditions. In co-culture with S. cerevisiae, K. lactis was completely inhibited and, at low aeration, died after 3 days; at high aeration this culture could survive but growth and lactose fermentation were only recovered after S. cerevisiae became stationary. To overcome the osmolarity and K. lactis' oxygen problems, the medium had to be diluted and a sequential fermentative process was designed in a STR-3l reactor. K. lactis was inoculated first and, with low aeration (0.13vvm), consumed all the lactose in 48h. Then S. cerevisiae was inoculated, consuming the total of the carob sugars, and producing ethanol in a fed-batch regime. The established co-culture with K. lactis increased S. cerevisiae ethanol tolerance. This fermentation process produced ethanol with good efficiency (80g/l final concentration and a conversion factor of 0.4g ethanol/g sugar), eliminating all the sugars of the mixed waste. These efficient fermentative results pointed to a new joint treatment of agro-industrial wastes which may be implemented successfully, with economic and environmental sustainability for a bioethanol industrial proposal.

  8. Growth kinetics and physiological behavior of co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis, fermenting carob sugars extracted with whey.

    PubMed

    Rodrigues, B; Lima-Costa, M E; Constantino, A; Raposo, S; Felizardo, C; Gonçalves, D; Fernandes, T; Dionísio, L; Peinado, J M

    2016-10-01

    Alcoholic fermentation of carob waste sugars (sucrose, glucose and fructose) extracted with cheese whey, by co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis has been analyzed. Growth and fermentation of S. cerevisiae in the carob-whey medium showed an inhibition of about 30% in comparison with water-extracted carob. The inhibition of K. lactis on carob-whey was greater (70%) when compared with the whey medium alone, due to osmolarity problems. Oxygen availability was a very important factor for K. lactis, influencing its fermentation performance. When K. lactis was grown alone on carob-whey medium, lactose was always consumed first, and glucose and fructose were consumed afterwards, only at high aeration conditions. In co-culture with S. cerevisiae, K. lactis was completely inhibited and, at low aeration, died after 3 days; at high aeration this culture could survive but growth and lactose fermentation were only recovered after S. cerevisiae became stationary. To overcome the osmolarity and K. lactis' oxygen problems, the medium had to be diluted and a sequential fermentative process was designed in a STR-3l reactor. K. lactis was inoculated first and, with low aeration (0.13vvm), consumed all the lactose in 48h. Then S. cerevisiae was inoculated, consuming the total of the carob sugars, and producing ethanol in a fed-batch regime. The established co-culture with K. lactis increased S. cerevisiae ethanol tolerance. This fermentation process produced ethanol with good efficiency (80g/l final concentration and a conversion factor of 0.4g ethanol/g sugar), eliminating all the sugars of the mixed waste. These efficient fermentative results pointed to a new joint treatment of agro-industrial wastes which may be implemented successfully, with economic and environmental sustainability for a bioethanol industrial proposal. PMID:27542743

  9. Selection of a Bifidobacterium animalis subsp. lactis Strain with a Decreased Ability To Produce Acetic Acid

    PubMed Central

    Margolles, Abelardo

    2012-01-01

    We have characterized a new strain, Bifidobacterium animalis subsp. lactis CECT 7953, obtained by random UV mutagenesis, which produces less acetic acid than the wild type (CECT 7954) in three different experimental settings: De Man-Rogosa-Sharpe broth without sodium acetate, resting cells, and skim milk. Genome sequencing revealed a single Phe-Ser substitution in the acetate kinase gene product that seems to be responsible for the strain's reduced acid production. Accordingly, acetate kinase specific activity was lower in the low acetate producer. Strain CECT 7953 produced less acetate, less ethanol, and more yoghourt-related volatile compounds in skim milk than the wild type did. Thus, CECT 7953 shows promising potential for the development of dairy products fermented exclusively by a bifidobacterial strain. PMID:22389372

  10. Kluyveromyces lactis killer system: analysis of cytoplasmic promoters of the linear plasmids.

    PubMed Central

    Schickel, J; Helmig, C; Meinhardt, F

    1996-01-01

    All of the 14 genes encoded by the cytoplasmic linear killer plasmids of Kluyveromyces lactis are preceded by upstream conserved sequences (UCSs), cis-acting elements involved in plasmid gene transcription. Using the bacterial glucose-dehydrogenase gene as a reporter, expression driven by seven cytoplasmic promoters was determined. The level of expression ranged from 0.5 to 6 nkat. The highest activity was displayed by UCS 6 of pGKL2 whereas the lowest level was obtained with UCS2 of pGKL2, all other values were in between. Sequences located 5' upstream the UCSs do not influence expression. As exemplified for UCS5 and UCS10, deletion led to an almost complete loss of expression. PMID:8657569

  11. Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

    PubMed Central

    Oxaran, Virginie; Ledue-Clier, Florence; Dieye, Yakhya; Herry, Jean-Marie; Péchoux, Christine; Meylheuc, Thierry; Briandet, Romain; Juillard, Vincent; Piard, Jean-Christophe

    2012-01-01

    The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used. PMID:23236417

  12. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate.

    PubMed

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  13. How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

    PubMed Central

    2011-01-01

    Background In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis. Results After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin

  14. Increased biomass yield of Lactococcus lactis during energetically limited growth and respiratory conditions.

    PubMed

    Koebmann, Brian; Blank, Lars Mathias; Solem, Christian; Petranovic, Dina; Nielsen, Lars K; Jensen, Peter Ruhdal

    2008-05-01

    Lactococcus lactis is known to be capable of respiration under aerobic conditions in the presence of haemin. In the present study the effect of respiration on ATP production during growth on different sugars was examined. With glucose as the sole carbon source, respiratory conditions in L. lactis MG1363 resulted in only a minor increase, 21%, in biomass yield. Since ATP production through substrate-level phosphorylation was essentially identical with and without respiration, the increased biomass yield was a result of energy-saving under respiratory conditions estimated to be 0.4 mol of ATP/mol of glucose. With maltose as the energy source, the increase in biomass yield amounted to 51% compared with an aerobic culture that lacked haemin. This higher ATP yield was obtained by redirecting pyruvate metabolism from lactate to acetate production, and from savings through respiration. However, even after subtracting these contributions, approx. 0.3 mol of ATP/mol of glucose remained unaccounted for. A similar response to respiratory conditions (0.2 mol of ATP/mol of glucose) was observed in a mutant that had a decreased glucose uptake rate during growth on glucose caused by disruption of the PTS(mannose) (glucose/mannose-specific phosphotransferase system). Amino acid catabolism could be excluded as the source of the additional ATP. Since mutants without a functional H+-ATPase produced less ATP under sugar starvation and respiratory conditions, the additional ATP yield appears to come partly from energy saved on proton pumping through the H+-ATPase due to respiration and partly from a reversed function of the H+-ATPase towards oxidative phosphorylation. These results may contribute to the design and implementation of carbon-efficient high-cell-density cultures of this industrially important species of bacterium.

  15. Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.

    PubMed

    Neves, A R; Ramos, A; Shearman, C; Gasson, M J; Almeida, J S; Santos, H

    2000-06-01

    The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.

  16. GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate

    PubMed Central

    Laroute, Valérie; Yasaro, Chonthicha; Narin, Waranya; Mazzoli, Roberto; Pessione, Enrica; Cocaign-Bousquet, Muriel; Loubière, Pascal

    2016-01-01

    Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes. PMID:27458444

  17. Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

    PubMed

    Torriani, S; Zapparoli, G; Dellaglio, F

    1999-10-01

    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

  18. Near infrared spectroscopy coupled with radial basis function neural network for at-line monitoring of Lactococcus lactis subsp. fermentation

    PubMed Central

    Liu, Yan; Lu, Chengyu; Meng, Qingfan; Lu, Jiahui; Fu, Yao; Liu, Botong; Zhou, Yongcan; Guo, Weiliang; Teng, Lesheng

    2015-01-01

    In our previous work, partial least squares (PLSs) were employed to develop the near infrared spectroscopy (NIRs) models for at-line (fast off-line) monitoring key parameters of Lactococcus lactis subsp. fermentation. In this study, radial basis function neural network (RBFNN) as a non-linear modeling method was investigated to develop NIRs models instead of PLS. A method named moving window radial basis function neural network (MWRBFNN) was applied to select the characteristic wavelength variables by using the degree approximation (Da) as criterion. Next, the RBFNN models with selected wavelength variables were optimized by selecting a suitable constant spread. Finally, the effective spectra pretreatment methods were selected by comparing the robustness of the optimum RBFNN models developed with pretreated spectra. The results demonstrated that the robustness of the optimal RBFNN models were better than the PLS models for at-line monitoring of glucose and pH of L. lactis subsp. fermentation. PMID:26858554

  19. Construction of a novel inducible expression vector for Lactococcus lactis M4 and Lactobacillus plantarum Pa21.

    PubMed

    Maidin, Mohd Shawal Thakib; Song, Adelene Ai-Lian; Jalilsood, Tannaz; Sieo, Chin Chin; Yusoff, Khatijah; Rahim, Raha Abdul

    2014-07-01

    A vector that drives the expression of the reporter gusA gene in both Lactobacillus plantarum and Lactococcus lactis was constructed in this study. This vector contained a newly characterized heat shock promoter (Phsp), amplified from an Enterococcus faecium plasmid, pAR6. Functionality and characterization of this promoter was initially performed by cloning Phsp into pNZ8008, a commercial lactococcal plasmid used for screening of putative promoters which utilizes gusA as a reporter. It was observed that Phsp was induced under heat, salinity and alkaline stresses or a combination of all three stresses. The newly characterized Phsp promoter was then used to construct a novel Lactobacillus vector, pAR1801 and its ability to express the gusA under stress-induced conditions was reproducible in both Lb. plantarum Pa21 and L. lactis M4 hosts.

  20. A peptidome-based phylogeny pipeline reveals differential peptides at the strain level within Bifidobacterium animalis subsp. lactis.

    PubMed

    Blanco-Míguez, Aitor; Gutiérrez-Jácome, Alberto; Fdez-Riverola, Florentino; Lourenço, Anália; Sánchez, Borja

    2016-12-01

    Bifidobacteria are gut commensal microorganisms belonging to the Actinobacteria group. Some specific strains of Bifidobacterium animalis subsp. lactis are used in functional foods as they are able to exert health-promoting effects in the human host. Due to the limited genetic variability within this subspecies, it is sometimes difficult for a manufacturer to properly track its strain once included in dairy products or functional foods. In this paper, we present a peptidome-based analysis in which the proteomes of a set of B. animalis subsp. lactis strains were digested in silico with human gut endopeptidases. The molecular masses were compared along all the strains to detect strain-specific peptides. These peptides may be interesting towards the development of methodologies for strain identification in the final product. PMID:27554155

  1. Evaluation of Phoenix Automated Microbiology System for detecting extended-spectrum beta-lactamases among chromosomal AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens.

    PubMed

    Park, Yeon-Joon; Yu, Jin Kyung; Lee, Seungok; Park, Jung-Jun; Oh, Eun-Jee

    2007-01-01

    We evaluated the BD Phoenix Extended-Spectrum beta-Lactamase (ESBL) detection test among chromosomal AmpC-producing Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens. The study was conducted on 72 non-repetitive ESBL producers (33 E. cloacae, 13 E. aerogenes, 14 C. freundii, and 12 S. marcescens) and 77 ESBL non-producers (33 E. cloacae, 9 E. aerogenes, 6 C. freundii, and 29 S. marcescens). The organisms were selected as suspected ESBL-producers based on the double disk synergy test and confirmed by PCR amplification of blaTEM-1, blaSHV-1, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9. The Phoenix ESBL test, using a 5-well confirmatory test and the BDXpert system, was evaluated. Of the 72 isolates identified as ESBL-producers based on the DDST, 46 isolates harbored CTX-M-type enzymes, 21 harbored TEM type enzymes, and 31 harbored SHV enzymes. The Phoenix system identified ESBL only in 15 isolates. Of the 77 ESBL non-producers, ths Phoenix system identified ESBL in 4 isolates, 3 of which were confirmed to be ESBL-producers. In this study, the Phoenix system was highly specific (76/77, 98.7%), and it identified 3 additional ESBL-producers that were not detected by DDST. However, the Phoenix system's sensitivity was very low (15/72, 20.8%). Considering the increasing prevalence of ESBL production among AmpC-producers, the BD Phoenix system could not be considered a reliable stand-alone ESBL detection method for the strains tested in our study.

  2. Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease.

    PubMed

    Farrugia, Mark A; Wang, Beibei; Feig, Michael; Hausinger, Robert P

    2015-10-20

    Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD/UreF/UreG complex, which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous, structurally characterized UreH/UreF/UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 Å predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-disrupting variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-down studies of cell-free extracts from tunnel-disrupting mutant cultures showed no loss of UreD interactions with urease or UreF/UreG. The nickel contents of urease samples enriched from activity-deficient cultures were decreased, while zinc and iron incorporation increased. Molecular dynamics simulations revealed size restrictions in the internal channels of the UreD variants. These findings support the role of a molecular tunnel in UreD as a direct facilitator of nickel transfer into urease, illustrating a new paradigm in active site metallocenter assembly.

  3. Mutational and Computational Evidence That a Nickel-Transfer Tunnel in UreD Is Used for Activation of Klebsiella aerogenes Urease.

    PubMed

    Farrugia, Mark A; Wang, Beibei; Feig, Michael; Hausinger, Robert P

    2015-10-20

    Nickel-containing urease from Klebsiella aerogenes requires four accessory proteins for proper active site metalation. The metallochaperone UreE delivers nickel to UreG, a GTPase that forms a UreD/UreF/UreG complex, which binds to urease apoprotein via UreD. Prior in silico analysis of the homologous, structurally characterized UreH/UreF/UreG complex from Helicobacter pylori identified a water tunnel originating at a likely nickel-binding motif in UreG, passing through UreF, and exiting UreH, suggestive of a role for the channel in providing the metal to urease apoprotein for its activation; however, no experimental support was reported for the significance of this tunnel. Here, specific variants were designed to disrupt a comparable 34.6 Å predicted internal tunnel, alternative channels, and surface sites for UreD. Cells producing a set of tunnel-disrupting variants of UreD exhibited greatly reduced urease specific activities, whereas other mutants had no appreciable effect on activity. Affinity pull-down studies of cell-free extracts from tunnel-disrupting mutant cultures showed no loss of UreD interactions with urease or UreF/UreG. The nickel contents of urease samples enriched from activity-deficient cultures were decreased, while zinc and iron incorporation increased. Molecular dynamics simulations revealed size restrictions in the internal channels of the UreD variants. These findings support the role of a molecular tunnel in UreD as a direct facilitator of nickel transfer into urease, illustrating a new paradigm in active site metallocenter assembly. PMID:26401965

  4. The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model.

    PubMed

    Guo, Shanguang; Yan, Weiwei; McDonough, Sean P; Lin, Nengfeng; Wu, Katherine J; He, Hongxuan; Xiang, Hua; Yang, Maosheng; Moreira, Maira Aparecida S; Chang, Yung-Fu

    2015-03-24

    Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2-3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI. PMID:25698490

  5. Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation.

    PubMed Central

    Crow, V L; Pritchard, G G

    1977-01-01

    The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo. PMID:17595

  6. Effect of bile on nisin-mediated antibacterial activity and the expression of nisin genes of Lactococcus lactis W8.

    PubMed

    Mitra, Suranjita; Mukhopadhyay, Bidhan Chandra; Chakrabartty, Pran Krishna; Biswas, Swadesh Ranjan

    2013-12-01

    The capability of Lactococcus lactis to produce nisin in the presence of bile in the intestinal environment remains an intriguing question. The aim of this study was to determine the effects of bile on production of nisin and the mRNA expression of nisin genes of L. lactis W8. The strain L. lactis W8 was grown on glucose in the absence and presence of bile (0.005-0.08 %) and the antibacterial activities of culture supernatants were determined. In culture with 0.035 % bile, the nisin activity was significantly reduced (400 AU/mL) within 5 h compared to that in the control without bile (2000 AU/mL), while growth of the cells was only slightly affected. In the presence of 0.07 % bile no nisin activity of the strain was manifested. Consistent with these results, mRNA expression of nisin-biosynthetic genes nisZ, nisRK, nisI, and nisF was down-regulated by 7.5-, 2.5-, 1.7-, and 6.0-fold, respectively in cells grown in the presence of bile (0.07 %) as compared to control culture without bile. The present study suggested that bile inhibited transcription of nisin genes. Nisin-production in intestine by orally administered L. lactis, thus, does not occur since complete inhibition of nisin-production by bile is observed at a concentration much lower than the physiological concentration (0.3 %) of bile present in the human intestine. The molecular mechanism underlying the bile-mediated inhibition of nisin genes remains to be elucidated. This is the first report on bile-mediated inhibition of nisin genes.

  7. Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

    PubMed Central

    Durmaz, Evelyn; Hu, Yan; Aroian, Raffi V.

    2015-01-01

    The Bacillus thuringiensis crystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) in Lactococcus lactis for potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production, cry5B was cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes in Lactococcus lysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strain L. lactis KP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates from L. lactis cultures expressing both Cry5B and tCry5B, in vivo challenges of Caenorhabditis elegans worms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly from L. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe. PMID:26682852

  8. Bifidobacterium animalis ssp. lactis CNCM-I2494 Restores Gut Barrier Permeability in Chronically Low-Grade Inflamed Mice

    PubMed Central

    Martín, Rebeca; Laval, Laure; Chain, Florian; Miquel, Sylvie; Natividad, Jane; Cherbuy, Claire; Sokol, Harry; Verdu, Elena F.; van Hylckama Vlieg, Johan; Bermudez-Humaran, Luis G.; Smokvina, Tamara; Langella, Philippe

    2016-01-01

    Growing evidence supports the efficacy of many probiotic strains in the management of gastrointestinal disorders associated with deregulated intestinal barrier function and/or structure. In particular, bifidobacteria have been studied for their efficacy to both prevent and treat a broad spectrum of animal and/or human gut disorders. The aim of the current work was thus to evaluate effects on intestinal barrier function of Bifidobacterium animalis ssp. lactis CNCM-I2494, a strain used in fermented dairy products. A chronic dinitrobenzene sulfonic acid (DNBS)-induced low-grade inflammation model causing gut dysfunction in mice was used in order to study markers of inflammation, intestinal permeability, and immune function in the presence of the bacterial strain. In this chronic low-grade inflammation mice model several parameters pointed out the absence of an over active inflammation process. However, gut permeability, lymphocyte populations, and colonic cytokines were found to be altered. B. animalis ssp. lactis CNCM-I2494 was able to protect barrier functions by restoring intestinal permeability, colonic goblet cell populations, and cytokine levels. Furthermore, tight junction (TJ) proteins levels were also measured by qRT-PCR showing the ability of this strain to specifically normalize the level of several TJ proteins, in particular for claudin-4. Finally, B. lactis strain counterbalanced CD4+ lymphocyte alterations in both spleen and mesenteric lymphoid nodes. It restores the Th1/Th2 ratio altered by the DNBS challenge (which locally augments CD4+ Th1 cells) by increasing the Th2 response as measured by the increase in the production of major representative Th2 cytokines (IL-4, IL-5, and IL-10). Altogether, these data suggest that B. animalis ssp. lactis CNCM-I2494 may efficiently prevent disorders associated with increased barrier permeability. PMID:27199937

  9. The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model.

    PubMed

    Guo, Shanguang; Yan, Weiwei; McDonough, Sean P; Lin, Nengfeng; Wu, Katherine J; He, Hongxuan; Xiang, Hua; Yang, Maosheng; Moreira, Maira Aparecida S; Chang, Yung-Fu

    2015-03-24

    Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2-3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI.

  10. Unraveling the Role of Surface Mucus-Binding Protein and Pili in Muco-Adhesion of Lactococcus lactis

    PubMed Central

    Duviau, Marie-Pierre; Meyrand, Mickael; Guérardel, Yann; Castelain, Mickaël; Loubière, Pascal; Chapot-Chartier, Marie-Pierre; Dague, Etienne; Mercier-Bonin, Muriel

    2013-01-01

    Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100−200 nm) and long distances (up to 600−800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants. PMID:24260308

  11. Bifidobacterium animalis ssp. lactis CNCM-I2494 Restores Gut Barrier Permeability in Chronically Low-Grade Inflamed Mice.

    PubMed

    Martín, Rebeca; Laval, Laure; Chain, Florian; Miquel, Sylvie; Natividad, Jane; Cherbuy, Claire; Sokol, Harry; Verdu, Elena F; van Hylckama Vlieg, Johan; Bermudez-Humaran, Luis G; Smokvina, Tamara; Langella, Philippe

    2016-01-01

    Growing evidence supports the efficacy of many probiotic strains in the management of gastrointestinal disorders associated with deregulated intestinal barrier function and/or structure. In particular, bifidobacteria have been studied for their efficacy to both prevent and treat a broad spectrum of animal and/or human gut disorders. The aim of the current work was thus to evaluate effects on intestinal barrier function of Bifidobacterium animalis ssp. lactis CNCM-I2494, a strain used in fermented dairy products. A chronic dinitrobenzene sulfonic acid (DNBS)-induced low-grade inflammation model causing gut dysfunction in mice was used in order to study markers of inflammation, intestinal permeability, and immune function in the presence of the bacterial strain. In this chronic low-grade inflammation mice model several parameters pointed out the absence of an over active inflammation process. However, gut permeability, lymphocyte populations, and colonic cytokines were found to be altered. B. animalis ssp. lactis CNCM-I2494 was able to protect barrier functions by restoring intestinal permeability, colonic goblet cell populations, and cytokine levels. Furthermore, tight junction (TJ) proteins levels were also measured by qRT-PCR showing the ability of this strain to specifically normalize the level of several TJ proteins, in particular for claudin-4. Finally, B. lactis strain counterbalanced CD4(+) lymphocyte alterations in both spleen and mesenteric lymphoid nodes. It restores the Th1/Th2 ratio altered by the DNBS challenge (which locally augments CD4(+) Th1 cells) by increasing the Th2 response as measured by the increase in the production of major representative Th2 cytokines (IL-4, IL-5, and IL-10). Altogether, these data suggest that B. animalis ssp. lactis CNCM-I2494 may efficiently prevent disorders associated with increased barrier permeability. PMID:27199937

  12. Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation.

    PubMed

    Yamaoka, Chizuru; Kurita, Osamu; Kubo, Tomoko

    2014-12-01

    The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.

  13. An autoselection system in recombinant Kluyveromyces lactis enhances cloned gene stability and provides freedom in medium selection.

    PubMed

    Hsieh, H P; Da Silva, N A

    1998-02-01

    An autoselection system for increasing plasmid stability in Kluyveromyces lactis, based on the blockage of the pyrimidine de novo and salvage pathways, was investigated. In a manner analogous to that used in Saccharomyces cerevisiae, a putative "fur 1" mutation was selected in a uraA K. lactis strain using 5-fluorouracil and 5-fluorocytosine plates. Survival of the mutant required expression of a plasmid-borne URA3 gene regardless of the culture medium employed, verifying the efficacy of this autoselection system in K. lactis. The expression of heterologous invertase, encoded by the S. cerevisiae SUC2 gene, was studied during long-term sequential batch cultures (70 generations) in complex yeast/peptone/glucose medium. The fur 1 mutant successfully retained the plasmid; invertase specific activity remained above 90% of the initial level. Furthermore, no mutation reversion was observed. In contrast, for the control non-fur 1 strain, only 4% of the cells retained the plasmid after 70 generations, and invertase specific activity dropped to less than 10% of the initial level. Experiments comparing growth and activity in different media indicated the potential for improving productivity through medium enrichment using this autoselection system.

  14. Codon and Propeptide Optimizations to Improve the Food-grade Expression of Bile Salt Hydrolase in Lactococcus lactis.

    PubMed

    Dong, Zixing; Zhang, Juan; Li, Huazhong; Du, Guocheng; Chen, Jian; Lee, Byonghoon

    2015-01-01

    To achieve the food-grade expression of bile salt hydrolase (BSH, EC 3.5.1.24) from Lactobacillus plantarum BBE7, the nisin controlled gene expression system (NICE), food-grade selection maker and signal peptide of Lactococcus lactis were used in this study. The open reading frame of BSH was optimized based on the codon bias of L. lactis, resulting in 12-fold and 9.5% increases in the intracellular and extracellular BSH activities, respectively. Three synthetic propeptides, LEISSTCDA (acidic), LGISSTCNA (neutral) and LKISSTCHA (basic) were also fused with signal peptide SPusp45 of vector pNZ8112 and introduced into the food-grade expression vector pNZ8149, respectively. Among these propeptides, acidic propeptide was effective in increasing the secretion efficiency and yield of BSH in recombinant bacteria, while neutral propeptide had no significant effect on the secretion of BSH. In contrast, basic propeptide strongly reduced the extracellular expression of BSH. By using codon optimization and the acidic propeptide together, the extracellular BSH activity was increased by 11.3%, reaching its maximum of 3.56 U/mg. To the best of our knowledge, this is the first report on the intracellular and extracellular expression of BSH using food-grade expression system, which would lay a solid foundation for large-scale production of BSH and other heterologous proteins in L. lactis. PMID:26059800

  15. The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

    PubMed Central

    Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

    2004-01-01

    We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

  16. Antagonistic action of cells of Lactobacillus delbrueckii subsp. lactis against pathogenic and spoilage microorganisms in fresh meat systems.

    PubMed

    Senne, M M; Gilliland, S E

    2003-03-01

    Cells of Lactobacillus delbrueckii subsp. lactis RM2-5 were added to various meat model systems that had been inoculated with Escherichia coli O157:H7 or Salmonella Typhimurium to determine whether these lactobacilli were antagonistic to the pathogens during storage at 5 degrees C. Experiments in which L. delbrueckii subsp. lactis RM2-5 was directly applied to the surfaces of beefsteaks resulted in significant (P < 0.05) reductions in the growth of psychrotrophs and coliforms plus a slight decrease in the numbers of E. coli O157:H7 over time relative to those for control samples to which no lactobacilli had been added. Experiments involving the direct application of L. delbrueckii subsp. lactis RM2-5 to the surfaces of freshly slaughtered beef and pork carcass samples inoculated with either E. coli O157:H7 or Salmonella Typhimurium showed significant (P < 0.05) declines in numbers of the pathogens as well as a reduction in the growth of psychrotrophs during storage at 5 degrees C for 6 days. The results of the experiments suggest that lactobacillus cultures have potential for use in an intervention technology for the control of foodborne pathogens, especially on the surfaces of beef and pork carcasses. The results of this study also suggest that an extension of the shelf life of meat can result from the decreased growth of psychrotrophic spoilage organisms. PMID:12636294

  17. Genome-Scale Genotype-Phenotype Matching of Two Lactococcus lactis Isolates from Plants Identifies Mechanisms of Adaptation to the Plant Niche▿ †

    PubMed Central

    Siezen, Roland J.; Starrenburg, Marjo J. C.; Boekhorst, Jos; Renckens, Bernadet; Molenaar, Douwe; van Hylckama Vlieg, Johan E. T.

    2008-01-01

    Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, but the species also occurs in various nondairy niches such as (fermented) plant material. Three genome sequences of L. lactis dairy strains (IL-1403, SK11, and MG1363) are publicly available. An extensive molecular and phenotypic diversity analysis was now performed on two L. lactis plant isolates. Diagnostic sequencing of their genomes resulted in over 2.5 Mb of sequence for each strain. A high synteny was found with the genome of L. lactis IL-1403, which was used as a template for contig mapping and locating deletions and insertions in the plant L. lactis genomes. Numerous genes were identified that do not have homologs in the published genome sequences of dairy L. lactis strains. Adaptation to growth on substrates derived from plant cell walls is evident from the presence of gene sets for the degradation of complex plant polymers such as xylan, arabinan, glucans, and fructans but also for the uptake and conversion of typical plant cell wall degradation products such as α-galactosides, β-glucosides, arabinose, xylose, galacturonate, glucuronate, and gluconate. Further niche-specific differences are found in genes for defense (nisin biosynthesis), stress response (nonribosomal peptide synthesis and various transporters), and exopolysaccharide biosynthesis, as well as the expected differences in various mobile elements such as prophages, plasmids, restriction-modification systems, and insertion sequence elements. Many of these genes were identified for the first time in Lactococcus lactis. In most cases good correspondence was found with the phenotypic characteristics of these two strains. PMID:18039825

  18. Kentucky Department for Natural Resources and Environmental Protection permit application for air contaminant source: SRC-I Demonstruation Plant, Newman, Kentucky. Appendix B. Best available control technology (BACT) proposals. [Demonstration plant at Newman, KY

    SciTech Connect

    Not Available

    1980-11-21

    The best available control technology (BACT) proposals for the following areas of the SRC-I demonstration plant are described: coal preparation and handling, SRC process and deashing, coke and liquid fuels (control of particles and hydrocarbon vapors), cryogenic systems and fuel gas purification (including sulfur recovery system and venting of gaseous wastes). (LTN)

  19. Profil bactériologique des sinusites maxillaires chroniques suppurées d'origine nasale de l'adulte au CHU Tokoin de Lomé

    PubMed Central

    Bathokédéou, Amana; Yaotse, Dagnra Anoumou; Essobozou, Pegbessou; Eyawelhon, Kpemissi

    2013-01-01

    Le but de cette étude était de déterminer les germes responsables des sinusites maxillaires chroniques et leur sensibilité aux antibiotiques usuels. Il s'agit d'une étude prospective menée sur 24 mois chez des patients reçus pour sinusite maxillaire chronique suppurée d'origine nasale. L'examen bactériologique était réalisé sur des prélèvements de pus obtenus par ponction des sinus. Sur les 100 prélèvements, 64 cultures (64%) étaient positives, dont 60 cultures étaient monomicrobiennes. Les germes les plus fréquemment isolés étaient le Streptococcus pneumoniae dans 20 cas (27.78%), le Staphylococcus aureus dans 15 cas (20.83%) et les streptocoques du groupe A dans 10 cas (13.89%). Les souches de Streptococcus pneumoniae étaient toutes sensible aux céphalosporines, aux quinolones, aux macrolides et à l'oxacilline. Les souches de Staphylococcus aureus avaient présenté 93.34% de résistance à la pénicilline G. Aucune souche n’était résistante à l'Oxacilline. Les streptocoques du groupe A avaient une sensibilité de 80 à 100% aux bétalactamines et aux macrolides, et 60% aux quinolones. L’écologie bactérienne des sinusites maxillaires chroniques suppurées de l'adulte est dominée par le Streptococcus pneumoniae et le Staphylococcus aureus. Ces germes sont sensibles aux quinolones et aux aminosides. PMID:24648861

  20. Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui, a Brazilian fermented, salted and dried meat product.

    PubMed

    Biscola, V; Todorov, S D; Capuano, V S C; Abriouel, H; Gálvez, A; Franco, B D G M

    2013-03-01

    A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0-10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui. PMID:23273471

  1. Proteinase PrtP impairs lactococcin LcnB activity in Lactococcus lactis BGMN1-501: new insights into bacteriocin regulation

    PubMed Central

    Vukotic, Goran; Mirkovic, Nemanja; Jovcic, Branko; Miljkovic, Marija; Strahinic, Ivana; Fira, Djordje; Radulovic, Zorica; Kojic, Milan

    2015-01-01

    Proteinases and bacteriocins are of great importance to the dairy industry, but their interactions have not been studied so far. Lactococcus lactis subsp. lactis BGMN1-5 is a natural isolate from homemade semi-hard cheese which produces two bacteriocins (Lactococcin B and LsbB), as well as proteinase PrtP. A medium-dependent increase in the bacteriocin LcnB activity of L. lactis BGMN1-501, a derivate of L. lactis subsp. lactis BGMN1-5, was shown to be accompanied by a decrease in its promoter activity. A similar effect of media components on gene expression was reported for proteinase PrtP, whose gene is co-localized on the same plasmid as the lcnB gene. Thus, the PrtP-LcnB interplay was investigated. Single gene knockout mutants were constructed with disrupted prtP or lcnB genes. PrtP- mutants showed higher bacteriocin activity that had lost its growth medium dependence, which was in contrast to the original strain. When LcnB from this mutant was combined with proteinase from the LcnB- mutant in vitro, its activity was rendered to the original level, suggesting that proteinase reduces bacteriocin activity. We propose a new model of medium dependent expression of these genes with regard to the effects of their interaction in vivo. PMID:25713574

  2. Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui, a Brazilian fermented, salted and dried meat product.

    PubMed

    Biscola, V; Todorov, S D; Capuano, V S C; Abriouel, H; Gálvez, A; Franco, B D G M

    2013-03-01

    A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0-10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.

  3. Bacteriophage Resistance of a ΔthyA Mutant of Lactococcus lactis Blocked in DNA Replication

    PubMed Central

    Pedersen, Martin B.; Jensen, Peter R.; Janzen, Thomas; Nilsson, Dan

    2002-01-01

    The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 ΔthyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher. PMID:12039762

  4. Evaluation of the performance of Torulaspora delbrueckii, Williopsis saturnus, and Kluyveromyces lactis in lychee wine fermentation.

    PubMed

    Chen, Dai; Yap, Zhi Yin; Liu, Shao-Quan

    2015-08-01

    This study evaluated the effects of three non-Saccharomyces yeasts, namely Torulaspora delbrueckii PRELUDE, Williopsis saturnus NCYC22, and Kluyveromyces lactis KL71 on lychee juice fermentation. The fermentation performance of these non-Saccharomyces yeasts was significantly different. T. delbrueckii PRELUDE had the fastest rate of growth and high sugar consumption. W. saturnus NCYC22 used the lowest amount of sugars, but consumed the highest amount of nitrogen. Correspondingly, strain PRELUDE produced the highest level of ethanol (7.6% v/v), followed by strain KL71 (3.4% v/v) and strain NCYC22 (0.8% v/v). Aroma character-impact terpenes and terpenoids could be partially retained in all lychee wines, with higher odour activity values (OAVs) of geraniol and citronellol in strain KL71. However, strain KL71 and strain NCYC22 over-produced ethyl acetate. Strain PRELUDE had a better ability to generate high levels of ethanol, isoamyl alcohol, 2-phenylethyl alcohol, ethyl octanoate, and ethyl decanoate and retained high OAVs of lychee aroma-character compounds cis-rose oxide (16.5) and linalool (3.5). Thus, it is deemed to be a promising non-Saccharomyces yeast for lychee wine fermentation.

  5. Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein.

    PubMed

    Xiao, Xue; Zhang, Changbin; Liu, Dajun; Bai, Weibin; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

    2016-01-01

    Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 10(10) Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract. PMID:27045906

  6. Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

    PubMed

    Nouaille, S; Bermúdez-Humarán, L G; Adel-Patient, K; Commissaire, J; Gruss, A; Wal, J M; Azevedo, V; Langella, P; Chatel, J M

    2005-03-01

    The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

  7. Effects of pH and Sugar on Acetoin Production from Citrate by Leuconostoc lactis.

    PubMed

    Cogan, T M; O'dowd, M; Mellerick, D

    1981-01-01

    The relationship between acetoin production and citrate utilization in Leuconostoc lactis NCW1 was studied. In a complex medium the organism utilized citrate at neutral pH (initial pH, 6.3) and at acid pH (initial pH, 4.5) but produced nine times more acetoin at the latter pH. In resting cells the utilization of citrate was optimum at pH 5.3. Production of acetoin as a function of citrate utilization increased as the pH decreased, and at pH 4.3 all of the citrate utilized was recovered as acetoin. Glucose (10 mM) and lactose (10 mM) markedly stimulated citrate utilization but totally inhibited acetoin production in glucose- and lactose-grown cells. Addition of glucose to cells actively metabolizing citrate caused an immediate increase in citrate uptake and a reduction in the level of acetoin. The apparent K(m) values of lactic dehydrogenase for pyruvate were 1.05, 0.25, and 0.15 mM at pH 7.5, 6.5, and 5.0, respectively. Several heterofermentation intermediates inhibited alpha-acetolactate synthetase and decarboxylase activities. The implications of these results in regulating acetoin formatin are discussed.

  8. Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein.

    PubMed

    Xiao, Xue; Zhang, Changbin; Liu, Dajun; Bai, Weibin; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

    2016-04-05

    Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 10(10) Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract.

  9. Evaluation of the performance of Torulaspora delbrueckii, Williopsis saturnus, and Kluyveromyces lactis in lychee wine fermentation.

    PubMed

    Chen, Dai; Yap, Zhi Yin; Liu, Shao-Quan

    2015-08-01

    This study evaluated the effects of three non-Saccharomyces yeasts, namely Torulaspora delbrueckii PRELUDE, Williopsis saturnus NCYC22, and Kluyveromyces lactis KL71 on lychee juice fermentation. The fermentation performance of these non-Saccharomyces yeasts was significantly different. T. delbrueckii PRELUDE had the fastest rate of growth and high sugar consumption. W. saturnus NCYC22 used the lowest amount of sugars, but consumed the highest amount of nitrogen. Correspondingly, strain PRELUDE produced the highest level of ethanol (7.6% v/v), followed by strain KL71 (3.4% v/v) and strain NCYC22 (0.8% v/v). Aroma character-impact terpenes and terpenoids could be partially retained in all lychee wines, with higher odour activity values (OAVs) of geraniol and citronellol in strain KL71. However, strain KL71 and strain NCYC22 over-produced ethyl acetate. Strain PRELUDE had a better ability to generate high levels of ethanol, isoamyl alcohol, 2-phenylethyl alcohol, ethyl octanoate, and ethyl decanoate and retained high OAVs of lychee aroma-character compounds cis-rose oxide (16.5) and linalool (3.5). Thus, it is deemed to be a promising non-Saccharomyces yeast for lychee wine fermentation. PMID:25955287

  10. NMR resonance assignments of the lantibiotic immunity protein NisI from Lactococcus lactis.

    PubMed

    Hacker, Carolin; Christ, Nina Alexandra; Duchardt-Ferner, Elke; Korn, Sophie; Berninger, Lucija; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2015-10-01

    The lantibiotic nisin is a small antimicrobial peptide which acts against a wide range of Gram-positive bacteria. Nisin-producing Lactococcus lactis strains express four genes for self-protection against their own antimicrobial compound. This immunity system consists of the lipoprotein NisI and the ABC transporter NisFEG. NisI is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Both the lipoprotein and the ABC transporter are needed for full immunity but the exact immunity mechanism is still unclear. To gain insights into the highly specific immunity mechanism of nisin producing strains on a structural level we present here the backbone resonance assignment of NisI (25.8 kDa) as well as the virtually complete (1)H,(15)N,(13)C chemical shift assignments for the isolated 12.7 kDa N-terminal and 14.6 kDa C-terminal domains of NisI.

  11. NMR resonance assignments of the lantibiotic immunity protein NisI from Lactococcus lactis.

    PubMed

    Hacker, Carolin; Christ, Nina Alexandra; Duchardt-Ferner, Elke; Korn, Sophie; Berninger, Lucija; Kötter, Peter; Entian, Karl-Dieter; Wöhnert, Jens

    2015-10-01

    The lantibiotic nisin is a small antimicrobial peptide which acts against a wide range of Gram-positive bacteria. Nisin-producing Lactococcus lactis strains express four genes for self-protection against their own antimicrobial compound. This immunity system consists of the lipoprotein NisI and the ABC transporter NisFEG. NisI is attached to the outside of the cytoplasmic membrane via a covalently linked diacylglycerol anchor. Both the lipoprotein and the ABC transporter are needed for full immunity but the exact immunity mechanism is still unclear. To gain insights into the highly specific immunity mechanism of nisin producing strains on a structural level we present here the backbone resonance assignment of NisI (25.8 kDa) as well as the virtually complete (1)H,(15)N,(13)C chemical shift assignments for the isolated 12.7 kDa N-terminal and 14.6 kDa C-terminal domains of NisI. PMID:25613223

  12. Identification of prophage genes expressed in lysogens of the Lactococcus lactis bacteriophage BK5-T.

    PubMed Central

    Boyce, J D; Davidson, B E; Hillier, A J

    1995-01-01

    Bacteriophage BK5-T is a small isometric-headed temperate phage that infects Lactococcus lactis subsp. cremoris. Northern (RNA) analysis of mRNA produced by lysogenic strains containing BK5-T prophage revealed four major BK5-T transcripts that are 0.8, 1.3, 1.8, and 1.8 kb in size and enabled a transcription map of the prophage genome to be prepared. The position and size of each transcript corresponded closely to the position and size of open reading frames predicted from the nucleotide sequence of BK5-T. Analysis of the transcripts suggested that one of them was derived from the gene encoding the BK5-T integrase and another was from the gene encoding the BK5-T homolog of the lambda cI repressor. Computer analysis of the nucleotide sequence upstream of the BK5-T cI homolog predicted the presence of a pair of divergent promoters and three inverted repeat sequences, features characteristic of temperature-phage immunity regions. By analogy with lambda, the three inverted repeat sequences could be binding sites for cI or Cro homologs and the two divergent promoters could initiate transcription through the BK5-T equivalents of cI and cro. PMID:8526524

  13. Survival Response and Rearrangement of Plasmid DNA of Lactococcus lactis during Long-Term Starvation

    PubMed Central

    Kim, Woojin S.; Park, Ji Hyeon; Ren, Jun; Su, Ping; Dunn, Noel W.

    2001-01-01

    The survival response of Lactococcus lactis during long-term starvation was investigated. The cells were cultured with different levels of glucose (the sole energy source) and either were kept in the resultant spent medium or transferred to fresh medium (without glucose) for up to 2 years. The survival of the cells during starvation was not dependent on the nature of transition phase, as expected, but on the nature of medium in which the cells were kept. The proliferation of cells, despite the apparent lack of glucose, could have been due to some cells being able to utilize the small amounts of peptides still present in the spent medium or to use energy sources provided by the breakup of dead cells. The 1- and 2-year-old cultures contained cells with vastly changed morphotypes. When these isolates were examined, it was revealed that the original plasmids present in the parent were rearranged in a certain way, and an entirely new plasmid was generated. Changes were also evident in the chromosomal DNA and in gene expression. Furthermore, all of the isolates exhibited a growth advantage relative to the parent cells when grown in energy-limiting media. When they were tested against different types of stresses, they exhibited a higher resistance against the bile salt and hydrogen peroxide stresses compared to the parent. Because of the similar changes observed in the 2-year-old isolates, a similar survival strategy may be operational in those cells that survive for that length of time. PMID:11571161

  14. Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein

    PubMed Central

    Xiao, Xue; Zhang, Changbin; Liu, Dajun; Bai, Weibin; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

    2016-01-01

    Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)–small molecule ubiquitin-like modifier protein (SUMO)–metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 1010 Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract. PMID:27045906

  15. Activity improvement of a Kluyveromyces lactis aldo-keto reductase KlAKR via rational design.

    PubMed

    Luo, Xi; Wang, Ya-Jun; Shen, Wei; Zheng, Yu-Guo

    2016-04-20

    Optically pure t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate ((R)-1b) is the key chiral precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. Wild-type aldo-keto reductase KlAKR from Kluyveromyces lactis has ideal diastereoselectivity toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a, dep>99.5%) but poor activity. A rational engineering was used to improve the KlAKR activity. Based on homology modeling and molecular docking, two amino acid residues (295 and 296) were selected as mutation sites, and two rounds of site-saturation mutagenesis were performed. Among the mutants, KlAKR-Y295W/W296L exhibited the highest catalytic efficiency (kcat/Km) toward 1a up to 12.37s(-1)mM(-1), which was 11.25-fold higher than that of wild-type KlAKR. Moreover, the majority of mutations have no negative impact on stereoselectivity. Using KlAKR-Y295W/W296L coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for cofactor regeneration, (R)-1b was accumulated up to 162.7mM with dep value above 99.5%. KlAKR-Y295W/W296L represents a robust tool for (R)-1b synthesis.

  16. Recovery and purification of a Kluyvermyces lactis β-galactosidase by Mixed Mode Chromatography.

    PubMed

    Lima, Micael de Andrade; de Freitas, Maria de Fátima Matos; Gonçalves, Luciana Rocha Barros; da Silva Junior, Ivanildo José

    2016-03-15

    Mixed Mode Chromatography (MMC) is a potential separation technique that allows simultaneous ionic and hydrophobic interactions between the adsorbent and the adsorbate. The aim of this work was to assess the recovery and purification of a Kluyveromyces lactis β-galactosidase employing MMC. Protein precipitation and dialysis were performed in order to concentrate the enzyme of interest and eliminate cell debris and other interferences inherent in the fermentation medium. The best conditions for both adsorption and desorption were attained by a non-factorial Central Composite Experimental Design and employed in the chromatographic runs with resin CAPTO MMC. Fermentation yielded mean values of total enzyme concentration of 0.44 mg/mL, enzymatic activity (employing lactose as a substrate) of 74 U/mL and specific activity of 168 U/mg. The Purification Factor (PF) obtained was of 1.17. After precipitation and dialysis, the subsequent chromatographic run resulted in recovery values ​​of 41.0 and 48.2% of total protein concentration and enzymatic activity, respectively. SDS-PAGE electrophoresis confirmed the purification evolution throughout the unit operations employed, attesting the feasibility of the technique to obtain enzymes with not only considerable degree of purity but also possessing high-added value.

  17. pH-Rate Profiles Support a General Base Mechanism for Galactokinase (Lactococcus lactis)

    PubMed Central

    Reinhardt, Laurie A.; Thoden, James B.; Peters, Greg S.; Holden, Hazel M.; Cleland, W.W.

    2013-01-01

    Galactokinase (GALK), a member the Leloir pathway for normal galactose metabolism, catalyzes the conversion of α-D-galactose to galactose-1-phosphate. For this investigation, we studied the kinetic mechanism and pH profiles of the enzyme from Lactococcus lactis. Our results show that the mechanism for its reaction is sequential in both directions. Mutant proteins D183A and D183N are inactive (<10,000 fold), supporting the role of Asp183 as a catalytic base that deprotonates the C-1 hydroxyl group of galactose. The pH–kcat profile of the forward reaction has a pKa of 6.9 ± 0.2 that likely is due to Asp183. The pH-kcat/KGal profile of the reverse reaction further substantiates this role as it is lacking a key pKa required for a direct proton transfer mechanism. The R36A and R36N mutant proteins show over 100-fold lower activity than that for the wild-type enzyme, thus suggesting that Arg36 lowers the pKa of the C-1 hydroxyl to facilitate deprotonation. PMID:23872454

  18. Bifidobacterium longum L-arabinose isomerase--overexpression in Lactococcus lactis, purification, and characterization.

    PubMed

    Salonen, Noora; Nyyssölä, Antti; Salonen, Kalle; Turunen, Ossi

    2012-09-01

    Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum L-AI was characterized using D-galactose and L-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0-6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K(m) value was 120 mM for L-arabinose and 590 mM for D-galactose. The V(max) was 42 U mg(-1) with L-arabinose and 7.7 U mg(-1) with D-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg(2+), Mn(2+)). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum L-AI as the catalyst at 35 °C, equilibrium yields of 36 % D-tagatose and 11 % L-ribulose with 1.67 M D-galactose and L-arabinose, respectively, as the substrates were reached.

  19. Production of xylitol from D-xylose by recombinant Lactococcus lactis.

    PubMed

    Nyyssölä, Antti; Pihlajaniemi, Anne; Palva, Airi; von Weymarn, Niklas; Leisola, Matti

    2005-07-21

    The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.

  20. Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic

    PubMed Central

    Mirkovic, Nemanja; Polovic, Natalija; Vukotic, Goran; Jovcic, Branko; Miljkovic, Marija; Radulovic, Zorica; Diep, Dzung B.

    2016-01-01

    Bacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins. Lactococcus lactis strains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (called lctLMG) was identified in LMG2081 but not in BGBM50. The lctLMG operon contains six open reading frames; the first three genes, lmgA, lmgM, and lmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes, lmgF, lmgE, and lmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that the lctLMG operon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization–time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid. PMID:26896142

  1. Kinetic modeling and sensitivity analysis of xylose metabolism in Lactococcus lactis IO-1.

    PubMed

    Oshiro, Mugihito; Shinto, Hideaki; Tashiro, Yukihiro; Miwa, Noriko; Sekiguchi, Tatsuya; Okamoto, Masahiro; Ishizaki, Ayaaki; Sonomoto, Kenji

    2009-11-01

    We proposed a kinetic simulation model of xylose metabolism in Lactococcus lactis IO-1 that describes the dynamic behavior of metabolites using the simulator WinBEST-KIT. This model was developed by comparing the experimental time-course data of metabolites in batch cultures grown in media with initial xylose concentrations of 20.3-57.8 g/l with corresponding calculated data. By introducing the terms of substrate activation, substrate inhibition, and product inhibition, the revised model showed a squared correlation coefficient (r2) of 0.929 between the experimental time-course of metabolites and the calculated data. Thus, the revised model is assumed to be one of the best candidates for kinetic simulation describing the dynamic behavior of metabolites. Sensitivity analysis revealed that pyruvate flux distribution is important for higher lactate production. To confirm the validity of our kinetic model, the results of the sensitivity analysis were compared with enzyme activities observed during increasing lactate production by adding natural rubber serum powder to the xylose medium. The experimental results on pyruvate flux distribution were consistent with the prediction by sensitivity analysis.

  2. Obligatory coupling between proton entry and the synthesis of adenosine 5'-triphosphate in Streptococcus lactis.

    PubMed Central

    Maloney, P C

    1977-01-01

    Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate. PMID:21165

  3. KNQ1, a Kluyveromyces lactis gene encoding a transmembrane protein, may be involved in iron homeostasis.

    PubMed

    Marchi, Emmanuela; Lodi, Tiziana; Donnini, Claudia

    2007-08-01

    The original purpose of the experiments described in this article was to identify, in the biotechnologically important yeast Kluyveromyces lactis, gene(s) that are potentially involved in oxidative protein folding within the endoplasmic reticulum (ER), which often represents a bottleneck for heterologous protein production. Because treatment with the membrane-permeable reducing agent dithiothreitol inhibits disulfide bond formation and mimics the reducing effect that the normal transit of folding proteins has in the ER environment, the strategy was to search for genes that conferred higher levels of resistance to dithiothreitol when present in multiple copies. We identified a gene (KNQ1) encoding a drug efflux permease for several toxic compounds that in multiple copies conferred increased dithiothreitol resistance. However, the KNQ1 product is not involved in the excretion of dithiothreitol or in recombinant protein secretion. We generated a knq1 null mutant, and showed that both overexpression and deletion of the KNQ1 gene resulted in increased resistance to dithiothreitol. KNQ1 amplification and deletion resulted in enhanced transcription of iron transport genes, suggesting, for the membrane-associated protein Knq1p, a new, unexpected role in iron homeostasis on which dithiothreitol tolerance may depend.

  4. Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells

    PubMed Central

    Andre, Guillaume; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre; Navet, Benjamine; Deghorain, Marie; Bernard, Elvis; Hols, Pascal; Dufrêne, Yves F.

    2010-01-01

    The spatial organization of peptidoglycan, the major constituent of bacterial cell-walls, is an important, yet still unsolved issue in microbiology. In this paper, we show that the combined use of atomic force microscopy and cell wall mutants is a powerful platform for probing the nanoscale architecture of cell wall peptidoglycan in living Gram-positive bacteria. Using topographic imaging, we found that Lactococcus lactis wild-type cells display a smooth, featureless surface morphology, whereas mutant strains lacking cell wall exopolysaccharides feature 25-nm-wide periodic bands running parallel to the short axis of the cell. In addition, we used single-molecule recognition imaging to show that parallel bands are made of peptidoglycan. Our data, obtained for the first time on living ovococci, argue for an architectural feature of the cell wall in the plane perpendicular to the long axis of the cell. The non-invasive live cell experiments presented here open new avenues for understanding the architecture and assembly of peptidoglycan in Gram-positive bacteria. PMID:20975688

  5. Mode of action of lactococcin R produced by Lactococcus lactis R.

    PubMed

    Yildirim, Zeliha; Yildirim, Metin; Johnson, Michael G

    2004-04-01

    We investigated the mode of action and factors affecting adsorption of lactoccocin R produced by Lactococcus lactis R. It was found that lactococcin R adsorbed to all Gram-positive but not to the Gram-negative bacteria tested and its adsorption was dependent on pH. It was observed that the binding of lactococcin R was prevented by anions of several salts (Cl-, PO4(-3)) and lipoteichoic acid. Pretreatments of sensitive cells and cell walls with detergents, organic solvents or enzymes did not reduce subsequent binding of lactococcin R. However, treatment of cell wall preparations with methanol:chloroform and hot 20% trichloroacetic acid (TCA) caused such walls to lose their ability to adsorb lactococcin R. Sensitive cells treated with lactococcin R lost high amounts of intracellular K+ ions, UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-glactopyranoside (ONPG). In addition, different lactococcin R concentrations (0-2560 AU/mL) decreased the colony counts of Listeria monocytogenes by 99% and also a reduction in the absorbance values. These results show that the mode of action of lactococcin R is bactericidal rather than bacteriostatic.

  6. Role of Snf1p in regulation of intracellular sorting of the lactose and galactose transporter Lac12p in Kluyveromyces lactis.

    PubMed

    Wiedemuth, Christian; Breunig, Karin D

    2005-04-01

    The protein kinase Snf1/AMPK plays a central role in carbon and energy homeostasis in yeasts and higher eukaryotes. To work out which aspects of the Snf1-controlled regulatory network are conserved in evolution, the Snf1 requirement in galactose metabolism was analyzed in the yeast Kluyveromyces lactis. Whereas galactose induction was only delayed, K. lactis snf1 mutants failed to accumulate the lactose/galactose H+ symporter Lac12p in the plasma membran,e as indicated by Lac12-green fluorescent protein fusions. In contrast to wild-type cells, the fusion protein was mostly intracellular in the mutant. Growth on galactose and galactose uptake could be restored by the KHT3 gene, which encodes a new transporter of the HXT subfamily of major facilitators These findings indicate a new role of Snf1p in regulation of sugar transport in K. lactis. PMID:15821131

  7. Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

    PubMed

    Robert, S; Van Huynegem, K; Gysemans, C; Mathieu, C; Rottiers, P; Steidler, L

    2015-01-01

    Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

  8. Oral Administration of Recombinant Lactococcus lactis Expressing HSP65 and Tandemly Repeated P277 Reduces the Incidence of Type I Diabetes in Non-Obese Diabetic Mice

    PubMed Central

    Ma, Yanjun; Liu, Jingjing; Hou, Jing; Dong, Yuankai; Lu, Yong; Jin, Liang; Cao, Rongyue; Li, Taiming; Wu, Jie

    2014-01-01

    Diabetes mellitus type 1 (DM1) is an autoimmune disease that gradually destroys insulin-producing beta-cells. We have previously reported that mucosal administration of fusion protein of HSP65 with tandem repeats of P277 (HSP65-6P277) can reduce the onset of DM1 in non-obese diabetic (NOD) mice. To deliver large amounts of the fusion protein and to enhance long-term immune tolerance effects, in the present study, we investigated the efficacy of using orally administrated L. lactis expressing HSP65-6P277 to reduce the incidence of DM1 in NOD mice. L. lactis strain NZ9000 was engineered to express HSP65-6P277 either constitutively or by nisin induction. After immunization via gavage with the recombinant L. lactis strains to groups of 4-week old female NOD mice for 36 weeks, we observed that oral administration of recombinant L. Lactis resulted in the prevention of hyperglycemia, improved glucose tolerance and reduced insulitis. Immunologic analysis showed that treatment with recombinant L. lactis induced HSP65- and P277- specific T cell immuno-tolerance, as well as antigen-specific proliferation of splenocytes. The results revealed that the DM1-preventing function was in part caused by a reduction in the pro-inflammatory cytokine IFN-γ and an increase in the anti-inflammatory cytokine IL-10. Orally administered recombinant L. lactis delivering HSP65-6P277 may be an effective therapeutic approach in preventing DM1. PMID:25157497

  9. Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase.

    PubMed

    Willemoës, Martin; Kilstrup, Mogens; Roepstorff, Peter; Hammer, Karin

    2002-08-01

    The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.

  10. Innate mechanisms for Bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats.

    PubMed

    Ruiz, Pedro A; Hoffmann, Micha; Szcesny, Silke; Blaut, Michael; Haller, Dirk

    2005-08-01

    Bifidobacteria comprise a dominant microbial population group in the human intestinal tract with purported beneficial health effects on the host. In this study, we characterized the molecular mechanisms for the initial interaction of probiotic Bifidobacterium lactis strain BB12 with native and intestinal epithelial cell (IEC) lines. We showed that B. lactis-monoassociated Fisher F344 rats transiently induce phosphorylation/activation of the NF-kappaB transcriptionally active subunit RelA and the mitogen-activated protein kinase (MAPK) p38 in native IEC at day 5 after initial bacterial colonization. In addition, Interleukin 6 (IL-6) gene expression was significantly increased at day 5, demonstrating the physiological relevance of transient transcription factor activation in IEC. In contrast, Bacteroides vulgatus-monoassociated Fisher rats revealed RelA but not p38 MAPK phosphorylation and failed to trigger significant IL-6 gene expression in native IEC. Moreover, we demonstrated that B. lactis triggers NF-kappaB RelA and p38 MAPK phosphorylation in IEC lines. Adenoviral delivery of mutant IKK-beta (Ad5dnIKKbeta) and inhibition of the p38 MAPK pathway through the pharmacological inhibitor SB203580 significantly blocked B. lactis-induced IL-6 gene expression in IEC, suggesting that B. lactis triggers NF-kappaB and MAPK signaling to induce gene expression in the intestinal epithelium. Regarding the mechanisms of bacteria epithelial cell cross-talk, B. lactis-induced IL-6 gene expression was completely inhibited in TLR2 deficient mouse embryogenic fibroblasts (MEF TLR2-/-) as well as TLR2DeltaTIR transfected Mode-K cells. In conclusion, we demonstrated that probiotic bacteria transiently trigger innate signal transduction and pro-inflammatory gene expression in the intestinal epithelium at early stages of bacterial colonization.

  11. Spontaneous deletion mutants of the Lactococcus lactis temperate bacteriophage BK5-T and localization of the BK5-T attP site.

    PubMed Central

    Boyce, J D; Davidson, B E; Hillier, A J

    1995-01-01

    Spontaneous deletion mutants of the temperate lactococcal bacteriophage BK5-T were obtained when the phage was grown vegetatively on the indicator strain Lactococcus lactis subsp. cremoris H2. One deletion mutant was unable to form stable lysogens, and analysis of this mutant led to the identification of the BK5-T attP site and the integrase gene (int). The core sequences of the BK5-T attP and host attB regions are conserved in a number of lactococcal phages and L. lactis strains. PMID:8526525

  12. Cloning, Production, and Functional Expression of the Bacteriocin Enterocin A, Produced by Enterococcus faecium T136, by the Yeasts Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, and Arxula adeninivorans

    PubMed Central

    Borrero, Juan; Kunze, Gotthard; Jiménez, Juan J.; Böer, Erik; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M.

    2012-01-01

    The bacteriocin enterocin A (EntA) produced by Enterococcus faecium T136 has been successfully cloned and produced by the yeasts Pichia pastoris X-33EA, Kluyveromyces lactis GG799EA, Hansenula polymorpha KL8-1EA, and Arxula adeninivorans G1212EA. Moreover, P. pastoris X-33EA and K. lactis GG799EA produced EntA in larger amounts and with higher antimicrobial and specific antimicrobial activities than the EntA produced by E. faecium T136. PMID:22685156

  13. Effect of in ovo administration of inulin and Lactococcus lactis on immune-related gene expression in broiler chickens.

    PubMed

    Płowiec, Arkadiusz; Sławińska, Anna; Siwek, Maria Z; Bednarczyk, Marek F

    2015-11-01

    OBJECTIVE To evaluate the effect of in ovo administration of inulin and Lactococcus lactis on immune-related gene expression in broiler chickens. ANIMALS 45 Ross broilers. PROCEDURES On day 12 of embryonic development, 360 eggs were equally allocated among 3 treatment groups and injected with 0.2 mL of a solution that contained 1.76 mg of inulin (prebiotic group) or 1.76 mg of inulin enriched with 1,000 CFUs of L lactis subsp lactis 2955 (synbiotic group), or they were injected with 0.2 mL of saline (0.9% NaCl) solution (control). At 1, 14, and 35 days after hatching, 5 male birds from each group were euthanized, and the spleen and cecal tonsils were harvested for determination of interleukin (IL)-4, IL-6, IL-8, IL-12p40, IL-18, cluster of differentiation 80, interferon-β, and interferon-γ expression by means of a reverse transcription quantitative PCR assay. Gene expressions in the cecal tonsils and spleens of chickens in the prebiotic and synbiotic groups were compared with those of control chickens at each tissue collection time. RESULTS Compared with control birds, immune-related gene expression was downregulated in birds in the prebiotic and synbiotic groups, and the magnitude of that downregulation was more pronounced in the cecal tonsils than in the spleen and increased with age. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that in ovo administration of a prebiotic or synbiotic to broilers was associated with downregulation of immune-related gene expression in the cecal tonsils and spleen. The magnitude of that downregulation increased with age and was most likely caused by stabilization of the gastrointestinal microbiota.

  14. Recombinant expressions of sweet plant protein mabinlin II in Escherichia coli and food-grade Lactococcus lactis.

    PubMed

    Gu, Wenliang; Xia, Qiyu; Yao, Jing; Fu, Shaoping; Guo, Jianchun; Hu, Xinwen

    2015-04-01

    Sweet plant proteins, which are safe, natural, low-calorie sweeteners, may be suitable replacements for sugars in the food and beverage industries. Mabinlin II, a sweet plant protein, shows the most pronounced heat stability and acid resistance of any of the six known types of plant sweet proteins. However, mabinlin II is difficult to extract from the Capparis masaikai plant, which is itself becoming increasingly scarce. This limits the use of naturally acquired mabinlin II. In this study, recombinant mabinlin II proteins were expressed and purified in Escherichia coli and in food-grade Lactococcus lactis. Recombinant mabinlin II proteins MBL-BH (containing the B-chains of mabinlin II downstream fused with His-tag) and MBL-ABH (containing the A- and B-chains of mabinlin II downstream fused with His-tag) were expressed in E. coli in the form of inclusion bodies. They were then purified and renatured. The refolded MBL-BH was found to be 100 times sweeter than sucrose by weight, but it was not heat-stable. Refolded MBL-ABH was neither sweet nor heat-stable. Recombinant mabinlin II proteins were secreted and expressed intracellularly in food-grade L. lactis, in which the concentrated cell samples and culture medium samples were detected using enzyme-linked immunosorbent assay and Western blotting analysis with anti-mabinlin II polyclonal antibody. This study demonstrated that the single B chain of mabinlin II has a sweet taste. The recombinant mabinlin II proteins have been successfully expressed in food-grade L. lactis, which is a crucial step in the production of mabinlin II through microorganism expression systems.

  15. N5-(1-carboxyethyl)-ornithine, a new amino acid from the intracellular pool of Streptococcus lactis.

    PubMed Central

    Thompson, J; Curtis, M A; Miller, S P

    1986-01-01

    Intracellular concentrations of amino acids were determined in cells of Streptococcus lactis 133 during growth in complex, spent, and chemically defined media. Glutamic and aspartic acids represented the major constituents of the amino acid pool. However, organisms grown in spent medium or in defined medium supplemented with ornithine also contained unusually high levels of two additional amino acids. One of these amino acids was ornithine. The second compound exhibited properties of a neutral amino acid by coelution with valine from the amino acid analyzer. The compound did not, however, comigrate with valine or any other standard amino acid by two-dimensional thin-layer chromatography. The unknown amino acid was purified by paper and thin-layer chromatography, and its molecular structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy. This new amino acid was shown to be N5-(1-carboxyethyl)-ornithine. The 14C-labeled compound was formed by cells of S. lactis 133 during growth in spent medium or defined medium containing [14C]ornithine. Formation of the derivative by resting cells required ornithine and the presence of a metabolizable sugar. N5-(1-Carboxyethyl)-ornithine was synthesized chemically from both poly-S-ornithine and (2S)-N2-carbobenzyloxy-ornithine as a 1:1 mixture of two diastereomers. The physical and chemical properties of the amino acid purified from S. lactis 133 were identical to those of one of the synthetic diastereomers. The bis-N-trifluoroacetyl-di-n-butyl esters of the natural and synthetic compounds generated identical gas chromatography-mass spectrometry spectra. A mechanism is suggested for the in vivo synthesis of N5-(1-carboxyethyl)-ornithine, and the possible functions of this new amino acid are discussed. Images PMID:3090017

  16. High yields of 2,3-butanediol and mannitol in Lactococcus lactis through engineering of NAD⁺ cofactor recycling.

    PubMed

    Gaspar, Paula; Neves, Ana Rute; Gasson, Michael J; Shearman, Claire A; Santos, Helena

    2011-10-01

    Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds.

  17. Stability of active prophages in industrial Lactococcus lactis strains in the presence of heat, acid, osmotic, oxidative and antibiotic stressors.

    PubMed

    Ho, Chun-Hoong; Stanton-Cook, Mitchell; Beatson, Scott A; Bansal, Nidhi; Turner, Mark S

    2016-03-01

    Lactococcus lactis is a starter bacterium commonly used in cheese making where it has an important role in acid-mediated curd formation as well as the development of flavour compounds. Industrial L. lactis strains can harbour one or more inducible prophages which when induced can affect cell growth and possibly lead to cell lysis. This is undesirable during growth and fermentation, but can beneficially lead to faster release of enzymes during cheese ripening. Lactococci can encounter multiple stress inducing conditions during the production of cheese, such as low and high temperatures, low pH, high osmotic pressure and long-term incubation. In this study, we tested the effect of these industrial stressors on prophage induction in two cheese making L. lactis subsp. cremoris strains (ASCC890049 and ASCC890310) as well as the laboratory strain L. lactis MG1363. Firstly, in order to identify inducible prophages in these strains we exposed them to the prophage inducing chemical mitomycin C (MMC) for 1 and 2h and then subjected the total genomic DNA to next-generation Illumina sequencing. Mapping of sequence reads back to the genome sequences revealed regions which contained a much higher fold coverage indicating DNA replication. These regions were amplified by up to 332-fold per cell (relative to the control tufA gene) and were identified as having similarities to different subgroups of P335 phages including MG-5, TP901-1, ul36.k1, bIL286, TP712 and BK5-T. Next, quantitative PCR was used to confirm the strong induction of prophages by MMC and then determine the copy number of the inducible prophages following exposure to various growth inhibitory levels of HCl, lactic acid, high temperature, NaCl, hydrogen peroxide and bacitracin. With the exception of a slight induction (2 to 4-fold) with hydrogen peroxide and long-term incubation after 21days in one industrial strain, none of the other stressors induced prophage DNA replication. These findings show that the repression

  18. Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis.

    PubMed

    Liu, W; Hansen, J N

    1990-08-01

    Nisin is a small gene-encoded antimicrobial protein produced by Lactococcus lactis that contains unusual dehydroalanine and dehydrobutyrine residues. The reactivity of these residues toward nucleophiles was explored by reacting nisin with a variety of mercaptans. The kinetics of reaction with 2-mercaptoethane-sulfonate and thioglycolate indicated that the reaction pathway includes a binding step. Reaction of nisin at high pH resulted in the formation of multimeric products, apparently as a result of intramolecular and intermolecular reactions between nucleophilic groups and the dehydro residues. One of the nucleophiles had a pKa of about 9.8. The unique vinyl protons of the dehydro residues that give readily identifiable proton nuclear magnetic resonances were used to observe the addition of nucleophiles to the dehydro moiety. After reaction with nucleophiles, nisin lost its antibiotic activity and no longer showed the dehydro resonances, indicating that the dehydro groups had been modified. The effect of pH on the solubility of nisin was determined; the solubility was quite high at low pH (57 mg/ml at pH 2) and was much lower at high pH (0.25 mg/ml at pH 8 to 12), as measured before significant pH-induced chemical modification had occurred. High-performance liquid chromatography on a C18 column was an effective technique for separating unmodified nisin from its reaction products. The cyanogen bromide cleavage products of nisin were about 90% less active toward inhibition of bacterial spore outgrowth than was native nisin. These results are consistent with earlier observations, which suggested that the dehydro residues of nisin have a role in the mechanism of antibiotic action, in which they act as electrophilic Michael acceptors toward nucleophiles in the cellular target. PMID:2119570

  19. The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis BB-12®

    PubMed Central

    Jungersen, Mikkel; Wind, Anette; Johansen, Eric; Christensen, Jeffrey E.; Stuer-Lauridsen, Birgitte; Eskesen, Dorte

    2014-01-01

    This review presents selected data on the probiotic strain Bifidobacterium animalis subsp. lactis BB-12® (BB-12®), which is the world’s most documented probiotic Bifidobacterium. It is described in more than 300 scientific publications out of which more than 130 are publications of human clinical studies. The complete genome sequence of BB-12® has been determined and published. BB-12® originates from Chr. Hansen’s collection of dairy cultures and has high stability in foods and as freeze dried powders. Strain characteristics and mechanisms of BB-12® have been established through extensive in vitro testing. BB-12® exhibits excellent gastric acid and bile tolerance; it contains bile salt hydrolase, and has strong mucus adherence properties, all valuable probiotic characteristics. Pathogen inhibition, barrier function enhancement, and immune interactions are mechanisms that all have been demonstrated for BB-12®. BB-12® has proven its beneficial health effect in numerous clinical studies within gastrointestinal health and immune function. Clinical studies have demonstrated survival of BB-12® through the gastrointestinal tract and BB-12® has been shown to support a healthy gastrointestinal microbiota. Furthermore, BB-12® has been shown to improve bowel function, to have a protective effect against diarrhea, and to reduce side effects of antibiotic treatment, such as antibiotic-associated diarrhea. In terms of immune function, clinical studies have shown that BB-12® increases the body’s resistance to common respiratory infections as well as reduces the incidence of acute respiratory tract infections. PMID:27682233

  20. The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis BB-12®

    PubMed Central

    Jungersen, Mikkel; Wind, Anette; Johansen, Eric; Christensen, Jeffrey E.; Stuer-Lauridsen, Birgitte; Eskesen, Dorte

    2014-01-01

    This review presents selected data on the probiotic strain Bifidobacterium animalis subsp. lactis BB-12® (BB-12®), which is the world’s most documented probiotic Bifidobacterium. It is described in more than 300 scientific publications out of which more than 130 are publications of human clinical studies. The complete genome sequence of BB-12® has been determined and published. BB-12® originates from Chr. Hansen’s collection of dairy cultures and has high stability in foods and as freeze dried powders. Strain characteristics and mechanisms of BB-12® have been established through extensive in vitro testing. BB-12® exhibits excellent gastric acid and bile tolerance; it contains bile salt hydrolase, and has strong mucus adherence properties, all valuable probiotic characteristics. Pathogen inhibition, barrier function enhancement, and immune interactions are mechanisms that all have been demonstrated for BB-12®. BB-12® has proven its beneficial health effect in numerous clinical studies within gastrointestinal health and immune function. Clinical studies have demonstrated survival of BB-12® through the gastrointestinal tract and BB-12® has been shown to support a healthy gastrointestinal microbiota. Furthermore, BB-12® has been shown to improve bowel function, to have a protective effect against diarrhea, and to reduce side effects of antibiotic treatment, such as antibiotic-associated diarrhea. In terms of immune function, clinical studies have shown that BB-12® increases the body’s resistance to common respiratory infections as well as reduces the incidence of acute respiratory tract infections.

  1. Dual mutations reveal interactions between components of oxidative phosphorylation in Kluyveromyces lactis.

    PubMed Central

    Clark-Walker, G D; Chen, X J

    2001-01-01

    Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for rho(0)-lethality has been identified by disruption of nuclear genes encoding electron transport and F(0)-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, Delta Psi, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F(1)F(0)-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or rho(0)-lethality can be suppressed by the atp2.1 mutation in the beta-subunit of F(1)-ATPase. Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F(1), allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain Delta Psi. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered to occur by mutant F(1) acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of rho(0)-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1. PMID:11729142

  2. Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis.

    PubMed

    Hill, C; Miller, L A; Klaenhammer, T R

    1990-11-01

    A number of host-encoded phage resistance mechanisms have been described in lactococci. However, the phage genome has not been exploited as a source of additional resistance determinants. A 4.5-kb BamHI-HindIII fragment of phage nck202.50 (phi 50) was subcloned in streptococcus-Escherichia coli shuttle plasmid pSA3 and introduced into Lactococcus lactis NCK203 and MG1363 by protoplast transformation. This cloned phage fragment directed a bacteriophage resistance phenotype designated Per (phage-encoded resistance). Both phi 50 and a distantly related phage, nck202.48 (phi 48), formed small plaques on strain NCK213 at a slightly reduced efficiency of plaquing on the Per+ host. The per locus was further reduced to a 1.4-kb fragment through in vitro deletion analysis. The 1.4-kb fragment was sequenced, and the Per phenotype was found to be associated with a ca. 500-bp region rich in direct and inverted repeats. We present evidence that the Per region contains a phage origin of replication which, in trans, may interfere with phage replication by titration of DNA polymerase or other essential replication factors. It was demonstrated that the Per+ phenotype is not a result of reduced adsorption or action of a restriction and modification system. Per+ activity was not detected against six independent phages which were previously shown to be sensitive to the Hsp+ mechanism. The mutually exclusive resistance mechanisms could be combined to confer resistance to both types of phages (Hsp resistant and Per resistant) in a single host. This is the first description in lactococci of a phage resistance phenotype, other than superinfection immunity, originating from a lactococcal phage genome.

  3. Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish.

    PubMed

    Yildirim, Z; Johnson, M G

    1998-04-01

    Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, alpha-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 degrees C for 15, 30 and 60 min, or 121 degrees C for 15 min. Lactococcin R remained active after storage at -20 and -70 degrees C for 3 months and after exposure to a pH of 2-9. The molecular weight of lactococcin R was about 2.5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml-1) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0.5% glucose, at 6.5-7.0 initial pH values, 30 degrees C temperature and 18-24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6-7 (95%). Crude lactococcin R at 1280 AU ml-1 was bactericidal, reducing colony counts of Listeria monocytogenes by 99.98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2.5 kDa vs 3.4 kDa, and in being sensitive to pepsin and alpha-chymotrypsin to which nisin is resistant. PMID:9633097

  4. Dual mutations reveal interactions between components of oxidative phosphorylation in Kluyveromyces lactis.

    PubMed

    Clark-Walker, G D; Chen, X J

    2001-11-01

    Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for rho(0)-lethality has been identified by disruption of nuclear genes encoding electron transport and F(0)-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, Delta Psi, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F(1)F(0)-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or rho(0)-lethality can be suppressed by the atp2.1 mutation in the beta-subunit of F(1)-ATPase. Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F(1), allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain Delta Psi. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered to occur by mutant F(1) acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of rho(0)-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1. PMID:11729142

  5. Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation.

    PubMed

    Murabito, Ettore; Verma, Malkhey; Bekker, Martijn; Bellomo, Domenico; Westerhoff, Hans V; Teusink, Bas; Steuer, Ralf

    2014-01-01

    Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models.

  6. Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis: Insights into Metabolic Regulation

    PubMed Central

    Murabito, Ettore; Verma, Malkhey; Bekker, Martijn; Bellomo, Domenico; Westerhoff, Hans V.; Teusink, Bas; Steuer, Ralf

    2014-01-01

    Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models. PMID:25268481

  7. Péritonites infectieuses en dialyse péritonéale continue ambulatoire au CHU de Rabat: profil bactériologique sur trois ans

    PubMed Central

    Lioussfi, Zineb; Rhou, Hakima; Ezzaitouni, Fatima; Ouzeddoun, Naima; Bayahia, Rabea; Benamar, Loubna

    2012-01-01

    Introduction La péritonite infectieuse (PI) est une des complications les plus sévères et les plus fréquentes de la dialyse péritonéale (DP). But: Déterminer le taux des PI et les germes en causes, et évaluer l’efficacité des protocoles thérapeutiques entrepris chez les patients traités par DP au CHU de Rabat. Méthodes Etude rétrospective effectuée en Septembre 2009 chez tous les patients traités par DP continue ambulatoire (DPCA) au CHU de Rabat depuis l’ouverture de l’unité de DP en Juillet 2006. Ont été inclus dans cette étude, tous les patients ayant fait une péritonite. Pour tous nos patients, nous avons relevé les données cliniques, biologiques et bactériologiques. Nous avons également recherché les causes des péritonites, le délai de survenue par rapport au début de la dialyse, et la durée moyenne de formation des patients. Résultats Au cours de la période de l’étude, 28 épisodes de PI sont survenus chez 19 patients dont la moyenne d’âge est de 46±16 (19-78) ans, avec une prédominance masculine (12 hommes/ 7 femmes). Le taux des PI dans notre unité de DP est de 21.07 mois-patients calculé par le RDPLF. Leur délai de survenue par rapport au début de la dialyse au centre est de 7.9 ±8 (1-29) mois. Lors de ces PI, les bactéries à Gram négatif (BGN) ont été retrouvées dans 55% des cas, contre uniquement 45% pour les Gram positifs. Conclusion La PI est une complication grave et redoutable de la DP. Le taux de PI dans notre centre de DPCA est de 21m-p ce qui correspond au taux acceptable définie par les sociétés internationales. Les germes les plus responsables des PI dans notre centre sont les BGN et la contamination semble être manu-portée se faisant essentiellement à partir de la flore environnementale et cutanée. PMID:22593777

  8. The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism.

    PubMed

    Burgess, Catherine M; Slotboom, Dirk Jan; Geertsma, Eric R; Duurkens, Ria H; Poolman, Bert; van Sinderen, Douwe

    2006-04-01

    This study describes the characterization of the riboflavin transport protein RibU in the lactic acid bacterium Lactococcus lactis subsp. cremoris NZ9000. RibU is predicted to contain five membrane-spanning segments and is a member of a novel transport protein family, not described in the Transport Classification Database. Transcriptional analysis revealed that ribU transcription is downregulated in response to riboflavin and flavin mononucleotide (FMN), presumably by means of the structurally conserved RFN (riboflavin) element located between the transcription start site and the start codon. An L. lactis strain carrying a mutated ribU gene exhibits altered transcriptional control of the riboflavin biosynthesis operon ribGBAH in response to riboflavin and FMN and does not consume riboflavin from its growth medium. Furthermore, it was shown that radiolabeled riboflavin is not taken up by the ribU mutant strain, in contrast to the wild-type strain, directly demonstrating the involvement of RibU in riboflavin uptake. FMN and the toxic riboflavin analogue roseoflavin were shown to inhibit riboflavin uptake and are likely to be RibU substrates. FMN transport by RibU is consistent with the observed transcriptional regulation of the ribGBAH operon by external FMN. The presented transport data are consistent with a uniport mechanism for riboflavin translocation and provide the first detailed molecular and functional analysis of a bacterial protein involved in riboflavin transport.

  9. Mesophilic Lactic Acid Bacteria Diversity Encountered in Brazilian Farms Producing Milk with Particular Interest in Lactococcus lactis Strains.

    PubMed

    Luiz, L M P; Chuat, V; Madec, M N; Araújo, E A; de Carvalho, A F; Valence, F

    2016-10-01

    The milk produced in regions with different traditions in Brazil is used for artisanal product production, which is characterized by different sensorial characteristics. This study aimed to identify the bacterial ecosystem of farms located in a traditional dairy region in the state of Minas Gerais and to characterize Lactococcus lactis strains, the species of interest in this study, using a multilocus sequence typing (MLST) protocol and pulsed-field gel electrophoresis (PFGE) technique. Samples were collected from raw milk and dairy environment from six farms. A total of 50 isolates were analyzed using 16S rRNA sequencing and species-specific PCR. Five genera were identified: Lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Staphylococcus, from ten different species. MLST (with six housekeeping genes) and PFGE (with SmaI endonuclease) were used for the characterization of 20 isolates of Lactococcus lactis from a dairy collection in this study. Both methods revealed a high clonal diversity of strains with a higher discriminatory level for PFGE (15 pulsotypes), compared to MLST (12 ST). This study contributes to the preservation of the Brazilian dairy heritage and provides insights into a part of the LAB population found in raw milk and dairy environment.

  10. Cell viability of microencapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and gastrointestinal tract simulation conditions.

    PubMed

    Shamekhi, Fatemeh; Shuhaimi, Mustafa; Ariff, Arbakariya; Manap, Yazid A

    2013-03-01

    The purpose of this study was to improve the survival of Bifidobacterium animalis subsp. lactis 10140 during freeze-drying process by microencapsulation, using a special pediatric prebiotics mixture (galactooligosaccharides and fructooligosaccharides). Probiotic microorganisms were encapsulated with a coat combination of prebiotics-calcium-alginate prior to freeze-drying. Both encapsulated and free cells were then freeze-dried in their optimized combinations of skim milk and prebiotics. Response surface methodology (RSM) was used to produce a coating combination as well as drying medium with the highest cell viability during freeze-drying. The optimum encapsulation composition was found to be 2.1 % Na-alginate, 2.9 % prebiotic, and 21.7 % glycerol. Maximum survival predicted by the model was 81.2 %. No significant (p > 0.05) difference between the predicted and experimental values verified the adequacy of final reduced models. The protection ability of encapsulation was then examined over 120 days of storage at 4 and 25 °C and exposure to a sequential model of infantile GIT conditions including both gastric conditions (pH 3.0 and 4.0, 90 min, 37 °C) and intestinal conditions (pH 7.5, 5 h, 37 °C). Significantly improved cell viability showed that microencapsulation of B. lactis 10140 with the prebiotics was successful in producing a stable symbiotic powdery nutraceutical.

  11. Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo.

    PubMed

    Cash, Darian D; Cohen-Zontag, Osnat; Kim, Nak-Kyoon; Shefer, Kinneret; Brown, Yogev; Ulyanov, Nikolai B; Tzfati, Yehuda; Feigon, Juli

    2013-07-01

    Telomerase is a ribonucleoprotein complex that extends the 3' ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C(+)•G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.

  12. In vivo and in vitro effects of rifampicin and streptolydigin on transcription of Kluyveromyces lactis in the presence of nystatin.

    PubMed Central

    Plevani, P; Badaracco, G; Marmiroli, N; Cassani, G

    1975-01-01

    Rifampicin and streptolydigin, if used in conjunction with nystatin, depress the growth of Kluyveromyces lactis. The incorporation of labeled leucine into protein is inhibited by nystatin whereas the incorporation of labeled uracil into RNA is inhibited by rifampicin in nystatin-treated cells. In order to study the mechanism of inhibition of RNA synthesis we purified by DEAE-Sephadex column chromatography four forms of RNA polymerase from K.lactis cells. The general properties of these enyzmes are similar to those of Saccharomyces cerevisiae and of other eukaryotic RNA polymerases. In particular, enzymes IA, IB and III are more active with poly[d(A-T)] template and Mn-2+ than with native or denatured calf thymus DNA. Enzyme II shows optimal activity with denatured calf thymus DNA and Mn2+. When challenged with native calf thymus DNA all enzymes prefer Mg-2+ as a divalent cation whereas with denatured calf thymus DNA all enzymes are more active with Mn-2+. Enzyme II is inhibited by lambda-amanitin but no enzyme is sensitive to rifampicin and streptolydigin. The inhibition of growth and uracil uptake observed when rifampicin is added to nystatin treated cells is probably not caused by a specific inhibition of transcription. PMID:1091916

  13. Generally recognized as safe (GRAS) Lactococcus lactis strains associated with Lippia sidoides Cham. are able to solubilize/mineralize phosphate.

    PubMed

    de Lacerda, Jackeline Rossetti Mateus; da Silva, Thais Freitas; Vollú, Renata Estebanez; Marques, Joana Montezano; Seldin, Lucy

    2016-01-01

    Eight strains isolated from the stems of Lippia sidoides were identified as belonging to Lactococcus lactis, a bacterial species considered as "generally recognized as safe". Their capacity to solubilize/mineralize phosphate was tested in vitro with different inorganic and organic phosphorus (P) sources. All strains were able to solubilize calcium phosphate as an inorganic P source, and the best result was observed with strain 003.41 which solubilized 31 % of this P source. Rock phosphate, a mined rock containing high amounts of phosphate bearing minerals, was solubilized by five strains. When calcium phytate was the organic P source used, the majority of the strains tested showed phosphate mineralization activity. Moreover, all strains were able to solubilize/mineralize phosphate from poultry litter, a complex P source containing inorganic and predominantly organic P. The presence of genes coding for phytase and alkaline phosphatase was searched within the strains studied. However, only gene sequences related to alkaline phosphatase (phoA and phoD) could be detected in the majority of the strains (excepting strain 006.29) with identities varying from 67 to 88 %. These results demonstrate for the first time the potential of L. lactis strains for phosphate solubilization/mineralization activity using a broad spectrum of P sources; therefore, they are of great importance for the future development of more safe bioinoculants with possible beneficial effects for agriculture.

  14. Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae.

    PubMed

    Sequeiros, Cynthia; Garcés, Marisa E; Vallejo, Marisol; Marguet, Emilio R; Olivera, Nelda L

    2015-04-01

    Bacteriocin-producing Lactococcus lactis TW34 was isolated from marine fish. TW34 bacteriocin inhibited the growth of the fish pathogen Lactococcus garvieae at 5 AU/ml (minimum inhibitory concentration), whereas the minimum bactericidal concentration was 10 AU/ml. Addition of TW34 bacteriocin to L. garvieae cultures resulted in a decrease of six orders of magnitude of viable cells counts demonstrating a bactericidal mode of action. The direct detection of the bacteriocin activity by Tricine-SDS-PAGE showed an active peptide with a molecular mass ca. 4.5 kDa. The analysis by MALDI-TOF-MS detected a strong signal at m/z 2,351.2 that corresponded to the nisin leader peptide mass without the initiating methionine, whose sequence STKDFNLDLVSVSKKDSGASPR was confirmed by MS/MS. Sequence analysis of nisin structural gene confirmed that L. lactis TW34 was a nisin Z producer. This nisin Z-producing strain with probiotic properties might be considered as an alternative in the prevention of lactococcosis, a global disease in aquaculture systems.

  15. Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese.

    PubMed

    Le Boucher, Clémentine; Gagnaire, Valérie; Briard-Bion, Valérie; Jardin, Julien; Maillard, Marie-Bernadette; Dervilly-Pinel, Gaud; Le Bizec, Bruno; Lortal, Sylvie; Jeanson, Sophie; Thierry, Anne

    2015-10-23

    In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening.

  16. Spatial Distribution of Lactococcus lactis Colonies Modulates the Production of Major Metabolites during the Ripening of a Model Cheese

    PubMed Central

    Le Boucher, Clémentine; Gagnaire, Valérie; Briard-Bion, Valérie; Jardin, Julien; Maillard, Marie-Bernadette; Dervilly-Pinel, Gaud; Le Bizec, Bruno; Lortal, Sylvie; Jeanson, Sophie

    2015-01-01

    In cheese, lactic acid bacteria are immobilized at the coagulation step and grow as colonies. The spatial distribution of bacterial colonies is characterized by the size and number of colonies for a given bacterial population within cheese. Our objective was to demonstrate that different spatial distributions, which lead to differences in the exchange surface between the colonies and the cheese matrix, can influence the ripening process. The strategy was to generate cheeses with the same growth and acidification of a Lactococcus lactis strain with two different spatial distributions, big and small colonies, to monitor the production of the major ripening metabolites, including sugars, organic acids, peptides, free amino acids, and volatile metabolites, over 1 month of ripening. The monitored metabolites were qualitatively the same for both cheeses, but many of them were more abundant in the small-colony cheeses than in the big-colony cheeses over 1 month of ripening. Therefore, the results obtained showed that two different spatial distributions of L. lactis modulated the ripening time course by generating moderate but significant differences in the rates of production or consumption for many of the metabolites commonly monitored throughout ripening. The present work further explores the immobilization of bacteria as colonies within cheese and highlights the consequences of this immobilization on cheese ripening. PMID:26497453

  17. Lytic infection of Lactococcus lactis by bacteriophages Tuc2009 and c2 triggers alternative transcriptional host responses.

    PubMed

    Ainsworth, Stuart; Zomer, Aldert; Mahony, Jennifer; van Sinderen, Douwe

    2013-08-01

    Here we present an entire temporal transcriptional profile of Lactococcus lactis subsp. cremoris UC509.9 undergoing lytic infection with two distinct bacteriophages, Tuc2009 and c2. Furthermore, corresponding high-resolution whole-phage genome tiling arrays of both bacteriophages were performed throughout lytic infection. Whole-genome microarrays performed at various time points postinfection demonstrated a rather modest impact on host transcription. The majority of changes in the host transcriptome occur during late infection stages; few changes in host gene transcription occur during the immediate and early infection stages. Alterations in the L. lactis UC509.9 transcriptome during lytic infection appear to be phage specific, with relatively few differentially transcribed genes shared between cells infected with Tuc2009 and those infected with c2. Despite the apparent lack of a coordinated general phage response, three themes common to both infections were noted: alternative transcription of genes involved in catabolic flux and energy production, differential transcription of genes involved in cell wall modification, and differential transcription of genes involved in the conversion of ribonucleotides to deoxyribonucleotides. The transcriptional profiles of both bacteriophages during lytic infection generally correlated with the findings of previous studies and allowed the confirmation of previously predicted promoter sequences. In addition, the host transcriptional response to lysogenization with Tuc2009 was monitored along with tiling array analysis of Tuc2009 in the lysogenic state. Analysis identified 44 host genes with altered transcription during lysogeny, 36 of which displayed levels of transcription significantly reduced from those for uninfected cells.

  18. Induction by glucose of an antimycin-insensitive, azide-sensitive respiration in the yeast Kluyveromyces lactis.

    PubMed

    Ferrero, I; Viola, A M; Goffeau, A

    1981-03-01

    Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (QO2 of 80 microliter O2 . h-1 . mg-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 microliter O2 . h-2 . mg-1 dry weight. Antimycin A inhibits the growth of K. lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced. It can be concluded that a minimal antimycin-insensitive QO2 is necessary for cellular growth when the normal respiratory pathway is not functional. The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 microM azide or 1 mM cyanide. These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements. A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells. The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.

  19. Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit.

    PubMed

    Zadravec, Petra; Marečková, Lucie; Petroková, Hana; Hodnik, Vesna; Perišić Nanut, Milica; Anderluh, Gregor; Štrukelj, Borut; Malý, Petr; Berlec, Aleš

    2016-01-01

    Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine. PMID:27606705

  20. Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

    PubMed Central

    Zadravec, Petra; Marečková, Lucie; Petroková, Hana; Hodnik, Vesna; Perišić Nanut, Milica; Anderluh, Gregor; Štrukelj, Borut; Malý, Petr; Berlec, Aleš

    2016-01-01

    Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine. PMID:27606705