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Sample records for ralstonia solanacearum strains

  1. Deciphering phenotypic diversity of Ralstonia solanacearum strains pathogenic to potato.

    PubMed

    Cellier, G; Prior, P

    2010-11-01

    Based on the phylotype classification, we questioned how genetically and phenotypically diverse strains of Ralstonia solanacearum pathogenic to potato may be. We studied 129 European and Mediterranean strains along with 57 reference strains known to cover genetic diversity in this species. Phylogeny analysis was done on endoglucanase gene sequences. Pathogenicity to potato, tomato, and eggplant was established at 24 to 30°C and 15 to 24°C, whereas tests on banana were conducted at 24 to 30°C. The ability to cause wilt on species of Solanaceae was shared by strains in all four phylotypes. Brown rot phylotypes IIB-1 and IIB-2 and phylotype IIB-27 established latent infections in banana, and Moko disease-causing phylotypes IIA-6, IIB-3, and IIB-4 were virulent to susceptible potato and tomato, addressing the question of host adaptation mechanisms, which may have undergone a similar bottleneck evolution. Cold-tolerance ability is only shared on species of Solanaceae among brown rot phylotype IIB-1, which gathered the majority of European and Mediterranean strains. We surveyed strain LNPV24.25 as the first report of an emerging phylotype IIB-4NPB strain in France. These findings showed that pathogenicity traits of genetically identified strains still need to be understood, especially in the perspective of post-genomics comparative analysis, to understand bacterial speciation in the R. solanacearum species complex.

  2. A multiplex PCR assay to detect and differentiate select agent strains of Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia solanacearum causes bacterial wilt in a variety of cash crops. R. solanacearum race 3 biovar 2 strains are considered select agents by the U.S. Government because they are not endemic to the U.S. and have the potential to cause brown rot disease in our potato production fields. Simple and...

  3. Genetic diversity and host range variation of Ralstonia solanacearum strains entering the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Each year, large volumes of plant propagative stock are imported into the United States; occasionally, Ralstonia solanacearum is found systemically infecting this plant material. In this study, 106 R. solanacearum strains were collected over a 10-year period from imported ornamental propagative sto...

  4. Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov.

    PubMed

    Safni, Irda; Cleenwerck, Ilse; De Vos, Paul; Fegan, Mark; Sly, Lindsay; Kappler, Ulrike

    2014-09-01

    The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S-23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA-DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain R 001(T) = LMG 10661(T) = DSM 7385(T)) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464(T) = LMG 27703(T) = DSM 27478(T)) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627(T

  5. Draft Genome Sequences of Nine Strains of Ralstonia solanacearum Differing in Virulence to Eggplant (Solanum melongena).

    PubMed

    Guinard, Jérémy; Vinatzer, Boris A; Poussier, Stéphane; Lefeuvre, Pierre; Wicker, Emmanuel

    2016-01-28

    Ralstonia solanacearum displays variability in its virulence to solanaceous crops. We report here the draft genome sequences of eight phylotype I strains and one phylotype III strain differing in virulence to the resistant eggplant genotype AG91-25. These data will allow the identification of virulence- and avirulence-related genes.

  6. Genetic diversity and host range variation of Ralstonia solanacearum strains entering North America.

    PubMed

    Norman, David J; Zapata, Mildred; Gabriel, Dean W; Duan, Y P; Yuen, Jeanne M F; Mangravita-Novo, Arianna; Donahoo, Ryan S

    2009-09-01

    Each year, large volumes of ornamental and food plant propagative stock are imported into the North America; occasionally, Ralstonia solanacearum is found systemically infecting this plant material. In this study, 107 new R. solanacearum strains were collected over a 10-year period from imported propagative stock and compared with 32 previously characterized R. solanacearum strains using repetitive polymerase chain reaction (rep-PCR) element (BOX, ERIC, and REP) primers. Additional strain comparisons were made by sequencing the endoglucanase and the cytochrome b561 genes. Using rep-PCR primers, populations could be distinguished by biovar and, to a limited extent, country of origin and original host. Similarity coefficients among rep-PCR clusters within biovars were relatively low in many cases, indicating that disease outbreaks over time may have been caused by different clonal populations. Similar population differentiations of R. solanacearum were obtained when comparing strain sequences using either the endoglucanase or cytochrome b561 genes. We found that most of the new biovar 1 strains of R. solanacearum entering the United States were genetically distinct from the biovar 1 strains currently found infecting vegetable production. These introduced biovar 1 strains also had a broader host range and could infect not only tomato, tobacco, and potato but also anthurium and pothos and cause symptoms on banana. All introductions into North America of race 3, biovar 2 strains in the last few years have been linked to geranium production and appeared to be clonal.

  7. Improved biovar test for Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test us...

  8. Tropical strains of Ralstonia solanacearum Outcompete race 3 biovar 2 strains at lowland tropical temperatures.

    PubMed

    Huerta, Alejandra I; Milling, Annett; Allen, Caitilyn

    2015-05-15

    Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.

  9. Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

    PubMed Central

    Huerta, Alejandra I.; Milling, Annett

    2015-01-01

    Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands. PMID:25769835

  10. Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential.

    PubMed

    Wicker, Emmanuel; Grassart, Laurence; Coranson-Beaudu, Régine; Mian, Danièle; Guilbaud, Caroline; Fegan, Mark; Prior, Philippe

    2007-11-01

    We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.

  11. [Serological characteristic of lipopolysaccharides of Ralstonia solanacearum].

    PubMed

    Hrytsaĭ, R V; Brovars'ka, O S; Zhytkevich, N V; Varbanets', L D

    2012-01-01

    By immunochemical investigations of eight strains of Ralstonia solanacearum six strains were attributed to four serogroups. Two of them are formed by pairs of R. solanacearum strains 4 and 526; 758 and 7954; two others are represented by single strains--TX1 Ta TS3, correspondingly. Antigenic structure of R. solanacearum 7954 O-polysaccharide unites antigenic epitopes of R. solanacearum strains 4, 35, 526, 749, however the absence of cross-reactivity does not permit uniting them into the same group. The latter, and also the fact that the antiserum to R. solanacearum 749 in the reaction with LPS of R solanacearum 526 forms two precipitation lines (while in the homological system it forms only one line) may be explained by differences in the component composition of heat-stable immunogens (which were used for antiserum obtaining), and also purified LPS which were utilized as antigens in immunochemical reactions.

  12. In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum

    PubMed Central

    Ailloud, Florent; Lowe, Tiffany M.; Robène, Isabelle; Cruveiller, Stéphane; Allen, Caitilyn

    2016-01-01

    Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes. PMID:26788428

  13. Genomic Analysis of Phylotype I Strain EP1 Reveals Substantial Divergence from Other Strains in the Ralstonia solanacearum Species Complex

    PubMed Central

    Li, Peng; Wang, Dechen; Yan, Jinli; Zhou, Jianuan; Deng, Yinyue; Jiang, Zide; Cao, Bihao; He, Zifu; Zhang, Lianhui

    2016-01-01

    Ralstonia solanacearum species complex is a devastating group of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the underlying genetic bases remain vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than reported genomes of other R. solanacearum strains. Both the chromosome and the mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, IV strains unveiled substantial genome rearrangements, insertions and deletions. Genome sequences are relatively conserved among the 4 phylotype I strains, but more divergent among strains of different phylotypes. Moreover, the strains exhibited considerable variations in their key virulence genes, including those encoding secretion systems and type III effectors. Our results provide valuable information for further elucidation of the genetic basis of diversified virulences and host range of R. solanacearum species. PMID:27833603

  14. Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles.

    PubMed

    Remenant, Benoît; de Cambiaire, Jean-Charles; Cellier, Gilles; Jacobs, Jonathan M; Mangenot, Sophie; Barbe, Valérie; Lajus, Aurélie; Vallenet, David; Medigue, Claudine; Fegan, Mark; Allen, Caitilyn; Prior, Philippe

    2011-01-01

    The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical

  15. Rhizocompetence and antagonistic activity towards genetically diverse Ralstonia solanacearum strains--an improved strategy for selecting biocontrol agents.

    PubMed

    Xue, Qing-Yun; Ding, Guo-Chun; Li, Shi-Mo; Yang, Yang; Lan, Cheng-Zhong; Guo, Jian-Hua; Smalla, Kornelia

    2013-02-01

    Bacterial wilt caused by Ralstonia solanacearum is a serious threat for agricultural production in China. Eight soil bacterial isolates with activity against R. solanacearum TM15 (biovar 3) were tested in this study for their in vitro activity towards ten genetically diverse R. solanacearum isolates from China. The results indicated that each antagonist showed remarkable differences in its ability to in vitro antagonize the ten different R. solanacearum strains. Strain XY21 (based on 16S rRNA gene sequencing affiliated to Serratia) was selected for further studies based on its in vitro antagonistic activity and its excellent rhizocompetence on tomato plants. Under greenhouse conditions XY21 mediated biocontrol of tomato wilt caused by seven different R. solanacearum strains ranged from 19 to 70 %. The establishment of XY21 and its effects on the bacterial community in the tomato rhizosphere were monitored by denaturing gradient gel electrophoresis of 16S rRNA gene fragments PCR-amplified from total community DNA. A positive correlation of the in vitro antagonistic activities of XY21 and the actual biocontrol efficacies towards seven genetically different R. solanacearum strains was found and further confirmed by the efficacy of XY21 in controlling bacterial wilt under field conditions.

  16. Draft Genome Sequence of Ralstonia solanacearum Strain Rs-T02, Which Represents the Most Prevalent Phylotype in Guangxi, China

    PubMed Central

    Zou, Chengwu; Wang, Kaihao; Meng, Jiaorong; Yuan, Gaoqing; Lin, Wei; Peng, Haowen

    2016-01-01

    Ralstonia solanacearum strain Rs-T02 was originally isolated from a bacterial wilt of tomato plant in Nanning City of Guangxi Province, China. It represents the most prevalent phylotype in Guangxi. Here, we present the draft genome sequence of this strain, which comprises 5,225 genes and 5,976,011 nucleotides with an average G+C content of 66.79%. There are 968 different genes between this isolate and the previously reported genome sequence of Ralstonia solanacearum GMl l000 (race l, biovar 3, phylotype I), and the genome sequence information of this isolate may be useful for comparative genomic studies to determine the genetic diversity in this species. PMID:27081126

  17. A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

    PubMed

    Cellier, Gilles; Moreau, Aurélie; Chabirand, Aude; Hostachy, Bruno; Ailloud, Florent; Prior, Philippe

    2015-01-01

    Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

  18. A Duplex PCR Assay for the Detection of Ralstonia solanacearum Phylotype II Strains in Musa spp.

    PubMed Central

    Cellier, Gilles; Moreau, Aurélie; Chabirand, Aude; Hostachy, Bruno; Ailloud, Florent; Prior, Philippe

    2015-01-01

    Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae. PMID:25811378

  19. Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution.

    PubMed

    Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2014-08-01

    The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I-IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15).

  20. Rapid differentiation of Ralstonia solanacearum avirulent and virulent strains by cell fractioning of an isolate using high performance liquid chromatography.

    PubMed

    Zheng, Xuefang; Zhu, Yujing; Liu, Bo; Yu, Qian; Lin, Naiquan

    2016-01-01

    Ralstonia solanacearum is one of the most destructive plant bacterial pathogens worldwide. The population dynamics and genetic stability are important issues, especially when an avirulent strain is used for biocontrol. In this study, we developed a rapid method to differentiate the virulent and avirulent strains of R. solanacearum and to predict the biocontrol efficiency of an avirulent strain using high performance liquid chromatography (HPLC). Three chromatographic peaks P1, P2 and P3 were observed on the HPLC spectra among 68 avirulent and 28 virulent R. solanacearum strains. Based on the HPLC peaks, 96 strains total were assigned to three categories. For avirulent strains, the intense peak is P1, while for virulent strains, P3 is the majority. Based on the HLPC spectra of R. solanacearum strains, a chromatography titer index (CTI) was established as CTIi = Si/(S1+S2+S3) × 100% (i represents an individual HPLC peak; S1, S2 and S3 represent peak areas of P1, P2 and P3, respectively). The avirulent strains had high values of CTI1 ranging from 63.6 to 100.0%, while the virulent strains displayed high values of CTI3 ranging from 90.2 to 100.0%. Biological inoculation studies of 68 avirulent strains revealed that the biocontrol efficacy was the best when CTI1 = 100%. The purity and genetic stability of R. solanacearum strains were confirmed in the P1 fraction of avirulent strain FJAT-1957 and P3 fraction of virulent strain FJAT-1925 after 30 generations of consecutive subculture. These results confirmed that fractioning by HPLC and their deduced CTI can be used for rapid and efficient evaluation and prediction of an isolate of R. solanacearum. To the best of our knowledge, this is the first report that HPLC fractioning can be used for rapid differentiation of virulent and avirulent strains of R. solanacearum.

  1. A novel, sensitive method to evaluate potato germplasm for bacterial wilt resistance using a luminescent Ralstonia solanacearum reporter strain.

    PubMed

    Cruz, Andrea Paola Zuluaga; Ferreira, Virginia; Pianzzola, María Julia; Siri, María Inés; Coll, Núria S; Valls, Marc

    2014-03-01

    Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.

  2. Cold Tolerance of some Ralstonia solanacearum strains, including Race3 Biovar2, is conferred in part by variation in cold shock gene cspD3.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia solanacearum race 3 biovar 2 (R3B2) strains are one of only 10 USDA Select Agents, a category of quarantined pathogens reserved for the most serious threats to U.S. plant industry. The threat of R3B2 strains was not considered to be likely due to race (these are poorly defined) or biovar ...

  3. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia solanacearum race 3 biovar 2 strains have the ability to cause brown rot of potato in temperate climates. Since these strains are not established in the U.S. and because of the potential risk they pose to the potato industry, the U.S. government has listed them as select agents. Cultivated...

  4. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China

    PubMed Central

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient. PMID:28197157

  5. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

    PubMed

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 10(2) CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.

  6. [Phytotoxic properties of Ralstonia solanacearum lipopolysaccharides].

    PubMed

    Hrytsaĭ, R V; Iakovleva, L M; Varbanets', L D

    2014-01-01

    The study is dedicated to research of phytotoxic properties of Ralstonia solanacearum lipopolysaccharides. This causative agent is one of the most dangerous among potato bacterial diseases. It is revealed that the inhibitory effect of LPS solution on seedlings germination is more noticeable on crops susceptible to brown rot. Maximal total phytotoxic properties have been shown by LPS from strains 35, 52b, TX1 and TS3, which were characterized by relatively low rhamnose content. Relative to the control plants LPS may diminish and some ones--increase the root length, height and weight of seedlings, subject to particular strain. But the stimulation revealed is minor.

  7. Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II.

    PubMed

    Albuquerque, Greecy M R; Santos, Liliana A; Felix, Kátia C S; Rollemberg, Christtianno L; Silva, Adriano M F; Souza, Elineide B; Cellier, Gilles; Prior, Philippe; Mariano, Rosa L R

    2014-11-01

    The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25.

  8. Control efficacy of an endophytic Bacillus amyloliquefaciens strain BZ6-1 against peanut bacterial Wilt, Ralstonia solanacearum.

    PubMed

    Wang, Xiaobing; Liang, Guobin

    2014-01-01

    We aimed to isolate and identify endophytic bacteria that might have efficacy against peanut bacterial wilt (BW) caused by Ralstonia solanacearum. Thirty-seven endophytic strains were isolated from healthy peanut plants in R. solanacearum-infested fields and eight showed antagonistic effects against R. solanacearum. Strain BZ6-1 with the highest antimicrobial activity was identified as Bacillus amyloliquefaciens based on morphology, biochemistry, and 16S rRNA analysis. Culture conditions of BZ6-1 were optimized using orthogonal test method and inhibitory zone diameter in dual culture plate assay reached 34.2 mm. Furthermore, main antimicrobial substances of surfactin and fengycin A homologues produced by BZ6-1 were analyzed by high performance liquid chromatography electrospray ionization tandem mass spectrometry. Finally, pot experiments were adopted to test the control efficiency of BZ6-1 against peanut BW. Disease incidence decreased significantly from 84.5% in the control to 12.1% with addition of 15 mL (10(8) cfu mL(-1)) culture broth for each seedling, suggesting the feasibility of strain BZ6-1 in the biological control of peanut plants BW.

  9. Phylogeny and population structure of brown rot- and Moko disease-causing strains of Ralstonia solanacearum phylotype II.

    PubMed

    Cellier, G; Remenant, B; Chiroleu, F; Lefeuvre, P; Prior, P

    2012-04-01

    The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools.

  10. Susceptibility of Geranium Cultivars (Pelargonium spp.) to Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sixty-one cultivars of geraniums including zonal, regal, ivy, and scented were tested for susceptibility to three strains of Ralstonia solanacearum: a Race 1 Biovar 1 (R1B1) strain P597 isolated from tomato in Florida, a R1B1 strain P673 obtained from pothos originating in Costa Rica, and a Race 3 B...

  11. Involvement of ralfuranones in the quorum sensing signaling pathway and virulence of Ralstonia solanacearum strain OE1-1.

    PubMed

    Mori, Yuka; Ishikawa, Shiho; Ohnishi, Hideyuki; Shimatani, Mika; Morikawa, Yukino; Hayashi, Kazusa; Ohnishi, Kouhei; Kiba, Akinori; Kai, Kenji; Hikichi, Yasufumi

    2017-01-23

    The soil-borne plant pathogenic Ralstonia solanacearum strain OE1-1 produces and secretes methyl 3-hydroxymyristate (3-OH MAME) as a quorum sensing (QS) signal, which contributes to its virulence. A global virulence regulator, PhcA, functioning through the QS system positively regulates the expression of ralA, which encodes furanone synthase, to produce aryl-furanone secondary metabolites, ralfuranones. A ralfuranone-deficient mutant (ΔralA) is weakly virulent when directly inoculated into tomato xylem vessels. To investigate the functions of ralfuranones, we analysed R. solanacearum transcriptome data generated by RNA sequencing technology. ΔralA expressed phcB, which is associated with 3-OH MAME production, and phcA at levels similar to those in strain OE1-1. Additionally, ΔralA exhibited downregulated expression of more than 90% of the QS-positively regulated genes, and upregulated expression of more than 75% of the QS-negatively regulated genes. These results suggest that ralfuranones affect the QS feedback loop. Ralfuranone supplementation restored the ability of ΔralA cells to aggregate. Additionally, ralfuranones A and B restored the swimming motility of ΔralA to wild-type levels. However, the application of exogenous ralfuranones did not affect the production of the major exopolysaccharide, EPS I, in ΔralA. Quantitative real-time PCR assays revealed that the deletion of ralA results in downregulated expression of vsrAD and vsrBC, which encode a sensor kinase and a response regulator, respectively, in the two-component regulatory systems that influence EPS I production. The application of ralfuranone B restored the expression of these two genes. Overall, our findings indicate that integrated signalling via ralfuranones influences the QS and virulence of R. solanacearum. This article is protected by copyright. All rights reserved.

  12. Microbial degradation of microcystin-LR by Ralstonia solanacearum.

    PubMed

    Zhang, M L; Yan, H; Pan, G

    2011-12-01

    A bacterial strain was isolated from Lake Dianchi (China) and its degradability and degradative pathways of the cyanobacterial toxin microcystin-LR (MC-LR) were studied. On the basis of morphological, physiological and biochemical tests, the strain was identified as Ralstonia solanacearum. The acute oral toxicity tests showed that Ralstonia solanacearum belongs to a non-toxic class. This bacterium degraded MC-LR at the rate of 9.4 mg/L per day, which was higher than those of the other bacterial strains reported in the literature. As for the degradative pathways, the results showed that the Adda-Arg peptide bond of MC-LR was initially hydrolysed by Ralstonia solanacearum to form a linear molecule as an intermediate. The intermediate product subsequently underwent a cyclisation reaction via dehydration to form a final product with a small peptide ring at one end of the molecule. These biodegradative pathways were different from those reported with other bacterial strains, suggesting that MC-LR may undergo different transformations, and different products were formed due to different compositions of bacteria present in natural lakes and reservoirs. These results suggest that there is a significant potential for Ralstonia solanacearum as a degrader for MC-LR removal from wastewater.

  13. Development and Comparison of TaqMan-Based Real-Time PCR Assays for Detection and Differentiation of Ralstonia solanacearum strains.

    PubMed

    Stulberg, Michael J; Rascoe, John; Li, Wenbin; Yan, Zonghe; Nakhla, Mark K; Huang, Qi

    2016-10-01

    Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.

  14. Molecular Diversity of Ralstonia solanacearum Isolated from Ginger in Hawaii.

    PubMed

    Yu, Q; Alvarez, A M; Moore, P H; Zee, F; Kim, M S; de Silva, A; Hepperly, P R; Ming, R

    2003-09-01

    ABSTRACT The genetic diversity of Ralstonia solanacearum strains isolated from ginger (Zingiber officinale) growing on the island of Hawaii was determined by analysis of amplified fragment length polymorphisms (AFLPs). Initially 28 strains of R. solanacearum collected from five host plant species worldwide were analyzed by AFLP. A second analysis was conducted on 55 R. solanacearum strains collected from three ginger farms along the Hamakua Coast of Hawaii, the principle area of ginger cultivation in the state. From the initial analysis, R. solanacearum strains from ginger in Hawaii showed a high degree of similarity at 0.853. In contrast, the average genetic similarity between R. solanacearum strains from heliconia and ginger was only 0.165, and strains from ginger showed little similarity with strains from all other hosts. The second analysis of 55 strains from ginger on different Hawaiian farms confirmed that they were distinct from race 1 strains from tomato. Strains from ginger also showed greater diversity among themselves in the second analysis, and the greatest diversity occurred among strains from a farm where ginger is frequently imported and maintained. Our results provide evidence that R. solanacearum strains from ginger in Hawaii are genetically distinct from local strains from tomato (race 1) and heliconia (race 2).

  15. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  16. Complete Genome Sequence of Ralstonia solanacearum FJAT-1458, a Potential Biocontrol Agent for Tomato Wilt

    PubMed Central

    Chen, Deju; Zhu, Yujing; Wang, Jieping; Chen, Zheng; Che, Jiamei; Zheng, Xuefang; Chen, Xiaoqiang

    2017-01-01

    ABSTRACT An avirulent strain of Ralstonia solanacearum FJAT-1458 was isolated from a living tomato. Here, we report the complete R. solanacearum FJAT-1458 genome sequence of 6,059,899 bp and 5,241 genes. This bacterial strain is a potential candidate as a biocontrol agent in the form of a plant vaccine for bacterial wilt. PMID:28385834

  17. A competitive index assay identifies several Ralstonia solanacearum type III effector mutant strains with reduced fitness in host plants.

    PubMed

    Macho, Alberto P; Guidot, Alice; Barberis, Patrick; Beuzón, Carmen R; Genin, Stéphane

    2010-09-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, is a soil bacterium which can naturally infect a wide range of host plants through the root system. Pathogenicity relies on a type III secretion system which delivers a large set of approximately 75 type III effectors (T3E) into plant cells. On several plants, pathogenicity assays based on quantification of wilting symptoms failed to detect a significant contribution of R. solanacearum T3E in this process, thus revealing the collective effect of T3E in pathogenesis. We developed a mixed infection-based method with R. solanacearum to monitor bacterial fitness in plant leaf tissues as a virulence assay. This accurate and sensitive assay provides evidence that growth defects can be detected for T3E mutants: we identified 12 genes contributing to bacterial fitness in eggplant leaves and 3 of them were also implicated in bacterial fitness on two other hosts, tomato and bean. Contribution to fitness of several T3E appears to be host specific, and we show that some known avirulence determinants such as popP2 or avrA do provide competitive advantages on some susceptible host plants. In addition, this assay revealed that the efe gene, which directs the production of ethylene by bacteria in plant tissues, and hdfB, involved in the biosynthesis of the secondary metabolite 3-hydroxy-oxindole, are also required for optimal growth in plant leaf tissues.

  18. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

  19. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

    PubMed Central

    Stulberg, Michael J.; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum. PMID:26426354

  20. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    DOE PAGES

    Stulberg, Michael J.; Huang, Qi

    2015-10-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regionsmore » of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.« less

  1. Ralstonia solanacearum RSc0411 (lptC) is a determinant for full virulence and has a strain-specific novel function in the T3SS activity.

    PubMed

    Yang, Wen-Chieh; Lin, Yu-Mei; Cheng, Yi-Sheng; Cheng, Chiu-Ping

    2013-06-01

    Previously, we have identified an avirulent Ralstonia solanacearum mutant carrying a transposon insertion in RSc0411, a gene homologous to the Escherichia coli LPS-transporting protein LptC. However, how the disruption of RSc0411 affects the bacterium-plant interactions and leads to decreased pathogenicity was not known. Here we show that the disruption of RSc0411 leads to pleiotropic defects, including reducing bacterial motility, biofilm formation, root attachment, rough-form LPS production and virulence in tomato and increasing membrane permeability. Disruption of the orthologous RSc0411 present in other R. solanacearum strains proves that most of these functions are conserved in the species. In contrast, trans-complementation analyses show that only RSc0411 orthologues from closely related bacteria can rescue the defects of the disruption mutant. These results enable us to propose a function for RSc0411, and for the clustered genes, in LPS biogenesis, and for the first time, to our knowledge, also a role of a gene from the DUF1239 gene family in bacterial pathogenicity. In addition and notably, the RSc0411 mutant displays a strain-specific phenotype for hypersensitive response (HR), in which the RSc0411 disruption impairs the HR caused by strain Pss190 but not that by strain Pss1308. Consistent with this strain-specific defect, the mutation clearly affects expression of the type III secretion system (T3SS) in Pss190 but not in other strains, suggesting that the HR-deficient phenotype of the RSc0411 mutant in Pss190 is due to impairment of the T3SS and thus RSc0411 has a strain-specific role in the T3SS activity of R. solanacearum.

  2. Roles of different forms of lipopolysaccharides in Ralstonia solanacearum pathogenesis.

    PubMed

    Li, Chien-Hui; Wang, Kuan-Chung; Hong, Yu-Hau; Chu, Tai-Hsiang; Chu, Yu-Ju; Chou, I-Chun; Lu, Der-Kang; Chen, Chiao-Yen; Yang, Wen-Chieh; Lin, Yu-Mei; Cheng, Chiu-Ping

    2014-05-01

    Lipopolysaccharides (LPS) are critical components for the fitness of most gram-negative bacteria. Ralstonia solanacearum causes a deadly wilting disease in many crops; however, the pathogenic roles of different forms of LPS and their pathways of biogenesis remain unknown. By screening for phage-resistant mutants of R. solanacearum Pss4, whose genome sequence is unavailable, mutants with various types of structural defects in LPS were isolated. Pathogenesis assays of the mutants revealed that production of rough LPS (R-LPS), which does not contain O-polysaccharides, was sufficient to cause necrosis on Nicotiana benthamiana and induce the hypersensitive response on N. tabacum. However, biosynthesis of smooth LPS (S-LPS), which contains O-polysaccharides, was required for bacterial proliferation at infection sites on N. benthamiana leaves and for proliferation and causing wilt on tomato. Complementation tests confirmed the involvement of the previously unidentified cluster RSc2201 to RSc2204 in the formation of R. solanacearum S-LPS. With these data and the availability of the annotated genomic sequence of strain GMI1000, certain loci involved in key steps of R. solanacearum LPS biosynthesis were identified. The strategy of this work could be useful for similar studies in other bacteria without available genome sequences.

  3. Transcriptomes of Ralstonia solanacearum during Root Colonization of Solanum commersonii

    PubMed Central

    Puigvert, Marina; Guarischi-Sousa, Rodrigo; Zuluaga, Paola; Coll, Núria S.; Macho, Alberto P.; Setubal, João C.; Valls, Marc

    2017-01-01

    Bacterial wilt of potatoes—also called brown rot—is a devastating disease caused by the vascular pathogen Ralstonia solanacearum that leads to significant yield loss. As in other plant-pathogen interactions, the first contacts established between the bacterium and the plant largely condition the disease outcome. Here, we studied the transcriptome of R. solanacearum UY031 early after infection in two accessions of the wild potato Solanum commersonii showing contrasting resistance to bacterial wilt. Total RNAs obtained from asymptomatic infected roots were deep sequenced and for 4,609 out of the 4,778 annotated genes in strain UY031 were recovered. Only 2 genes were differentially-expressed between the resistant and the susceptible plant accessions, suggesting that the bacterial component plays a minor role in the establishment of disease. On the contrary, 422 genes were differentially expressed (DE) in planta compared to growth on a synthetic rich medium. Only 73 of these genes had been previously identified as DE in a transcriptome of R. solanacearum extracted from infected tomato xylem vessels. Virulence determinants such as the Type Three Secretion System (T3SS) and its effector proteins, motility structures, and reactive oxygen species (ROS) detoxifying enzymes were induced during infection of S. commersonii. On the contrary, metabolic activities were mostly repressed during early root colonization, with the notable exception of nitrogen metabolism, sulfate reduction and phosphate uptake. Several of the R. solanacearum genes identified as significantly up-regulated during infection had not been previously described as virulence factors. This is the first report describing the R. solanacearum transcriptome directly obtained from infected tissue and also the first to analyze bacterial gene expression in the roots, where plant infection takes place. We also demonstrate that the bacterial transcriptome in planta can be studied when pathogen numbers are low by

  4. Genomic characterization of Ralstonia solanacearum phage ϕRS138 of the family Siphoviridae.

    PubMed

    Van Truong Thi, Bich; Pham Khanh, Nguyen Huan; Namikawa, Ryuta; Miki, Kaito; Kondo, Akihiro; Dang Thi, Phuong Thao; Kamei, Kaeko

    2016-02-01

    ϕRS138, a bacteriophage of the family Siphoviridae that lyses Ralstonia solanacearum, was isolated. The genomic DNA of ϕRS138 was 41,941 bp long with a GC content of 65.1 % and contained 56 putative open reading frames. The ϕRS138 genome could be divided into three regions based on similarities to other genomes: (1) a region containing genes encoding a putative transcriptional regulator and an integrase, similar to the prophage genes in Ralstonia solanacearum K60-1; (2) a region encoding proteins related to structural modules and virion morphogenesis, similar to genes in the Pseudomonas phages of the family Siphoviridae; and (3) a region highly similar to the genomes of other Ralstonia solanacearum strains.

  5. Antagonistic activity and mechanisms of Bacillus subtilis SB1 against Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, showed a broad-spectrum of antimicrobial activity in vitro experiments. In addition to Ralstonia solanacearum, strain SB1 inhibited the growth of many other plant pathogens, including Fusarium oxysporum, Botrytis cinerea, Phytoph...

  6. Response of tomato wilt pathogen Ralstonia solanacearum to the volatile organic compounds produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9

    PubMed Central

    Raza, Waseem; Ling, Ning; Yang, Liudong; Huang, Qiwei; Shen, Qirong

    2016-01-01

    It is important to study the response of plant pathogens to the antibiosis traits of biocontrol microbes to design the efficient biocontrol strategies. In this study, we evaluated the role of volatile organic compounds (VOCs) produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum (RS). The VOCs of SQR-9 significantly inhibited the growth of RS on agar medium and in soil. In addition, the VOCs significantly inhibited the motility traits, production of antioxidant enzymes and exopolysaccharides, biofilm formation and tomato root colonization by RS. The strain SQR-9 produced 22 VOCs, but only nine VOCs showed 1–11% antibacterial activity against RS in their corresponding amounts; however, the consortium of all VOCs showed 70% growth inhibition of RS. The proteomics analysis showed that the VOCs of SQR-9 downregulated RS proteins related to the antioxidant activity, virulence, carbohydrate and amino acid metabolism, protein folding and translation, while the proteins involved in the ABC transporter system, amino acid synthesis, detoxification of aldehydes and ketones, methylation, protein translation and folding, and energy transfer were upregulated. This study describes the significance and effectiveness of VOCs produced by a biocontrol strain against tomato wilt pathogen. PMID:27103342

  7. Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)

    PubMed Central

    Moissenet, Didier; Bidet, Philippe; Garbarg-Chenon, Antoine; Arlet, Guillaume; Vu-Thien, Hoang

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNAIle and tRNAAla genes, which are identical to genes described for R. pickettii and R. solanacearum. PMID:11136807

  8. Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

    PubMed

    Moissenet, D; Bidet, P; Garbarg-Chenon, A; Arlet, G; Vu-Thien, H

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

  9. A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex.

    PubMed

    Ravelomanantsoa, Santatra; Robène, Isabelle; Chiroleu, Frédéric; Guérin, Fabien; Poussier, Stéphane; Pruvost, Olivier; Prior, Philippe

    2016-01-01

    Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC

  10. A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex

    PubMed Central

    Ravelomanantsoa, Santatra; Robène, Isabelle; Chiroleu, Frédéric; Guérin, Fabien; Poussier, Stéphane; Pruvost, Olivier

    2016-01-01

    Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC

  11. Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2013-01-01

    Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg(2+)-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum.

  12. Comparison between Solanum torvum Sw. and S. melongena L. after Ralstonia solanacearum inoculation.

    PubMed

    Aribaud, M; Noirot, M; Fock-Bastide, I; Vaniet, S; Kodja, H

    2014-09-01

    Bacterial wilt, caused by Ralstonia solanacearum, is one of the most devastating plant diseases, affecting some economically important Solanaceae crops. In contrast, Solanum torvum, also known as wild eggplant, does not wilt when infested with R. solanacearum. In order to describe the mechanism underlying the response of S. torvum, it was compared with the cultivated eggplant, S. melongena, when both were infected with the same R. solanacearum strain. No wilting occurred in S. torvum, although the bacteria colonised roots and stems in both species within the first 24 h. There were marked differences beyond 24 h, consisting of high bacterial mortality in S. torvum. Using the calli model, our investigations revealed an increase in cell wall monoamine oxidase activity in S. torvum after R. solanacearum inoculation, which did not occur in S. melongena.

  13. Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum.

    PubMed

    Pauly, Julia; Spiteller, Dieter; Linz, Jeanine; Jacobs, Jonathan; Allen, Caitilyn; Nett, Markus; Hoffmeister, Dirk

    2013-11-04

    Ralfuranones are aryl-substituted furanone secondary metabolites of the Gram-negative plant pathogen Ralstonia solanacearum. New sulfur-containing ralfuranone derivatives were identified, including the methyl thioether-containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione.

  14. A computer program for fast and easy typing of partial endoglucanase gene sequence into phylotypes and sequevars 1&2 (select agents) of Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phytopathogen Ralstonia solanacearum is a species complex that contains a subset of strains that are quarantined or select agent pathogens. An unidentified R. solanacearum strain is considered a select agent in the US until proven otherwise, which can be done by phylogenetic analysis of a partia...

  15. A computer program for fast and easy typing of a partial endoglucanase gene sequence into genospecies and sequevars 1&2 of the Ralstonia solanacearum species complex.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2016-04-01

    The phytopathogen Ralstonia solanacearum is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the R. solanacearum species complex strains have been reclassified into three genospecies: R. solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii. An unidentified R. solanacearum strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). Currently, sequevars of R. solanacearum species complex strains can only be determined by phylogenetic analysis of a partial endoglucanase (egl) sequence of approximately 700-bp in length. Such analysis, however, requires expert knowledge to properly trim the sequence, to include the correct reference strains, and to interpret the results. By comparing GenBank egl sequences of representative R. solanacearum species-complex strains, we identified genospecies- and sequevar 1 and 2-specific single nucleotide polymorphisms (SNPs). We also designed primers to amplify a shorter, 526-bp, egl fragment from R. solanacearum species complex strains for easy sequencing of the amplicon, and to facilitate direct and specific amplification of egl from R. solanacearum-infected plant samples without the need of bacterial isolation. We wrote a computer program (Ralstonia solanacearum typing program) that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. The program correctly typed all 371 tested egl sequences with known sequevars, obtained either from GenBank or through personal communication. Additionally, the program successfully typed 25 R. solanacearum strains in our

  16. Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031.

    PubMed

    Guarischi-Sousa, Rodrigo; Puigvert, Marina; Coll, Núria S; Siri, María Inés; Pianzzola, María Julia; Valls, Marc; Setubal, João C

    2016-01-01

    Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain's virulence.

  17. Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2016-01-01

    Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.

  18. Application of variable-number tandem-repeat typing to discriminate Ralstonia solanacearum strains associated with English watercourses and disease outbreaks.

    PubMed

    Parkinson, Neil; Bryant, Ruth; Bew, Janice; Conyers, Christine; Stones, Robert; Alcock, Michael; Elphinstone, John

    2013-10-01

    Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.

  19. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    SciTech Connect

    Stulberg, Michael J.; Huang, Qi

    2015-10-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

  20. Opening the Ralstonia solanacearum type III effector tool box: insights into host cell subversion mechanisms.

    PubMed

    Deslandes, Laurent; Genin, Stephane

    2014-08-01

    Effectors delivered to host cells by the Type III secretion system are essential to Ralstonia solanacearum pathogenicity, as in several other plant pathogenic bacteria. The establishment of exhaustive effector repertoires in multiple R. solanacearum strains drew a first picture of the evolutionary dynamics of the pathogen effector suites. Effector repertoires are diversified, with a core of 20-30 effectors present in most of the strains and the obtention of mutants lacking one or more effector genes revealed the functional overlap among this effector network. Recent functional studies have provided insights into the ability of single effectors to manipulate the host proteasome, elicit cell death, trigger the expression of plant genes, and/or display biochemical activities on plant protein targets.

  1. Involvement of ralfuranone production in the virulence of Ralstonia solanacearum OE1-1.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Mori, Yuka; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2014-11-24

    Ralstonia solanacearum causes a destructive disease called "bacterial wilt" in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell-density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J-L, new PhcA-regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1-1. Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1-1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1-1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1-1.

  2. Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum

    PubMed Central

    Kong, Hyun Gi; Jo, Eun Jeong; Choi, Hye Kyung; Khan, Raees; Lee, Seon-Woo

    2016-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that infects more than 200 plant species. Its broad host range and long-term survival under different environmental stress conditions suggest that it uses a variety of mechanisms to protect itself against various types of biotic and abiotic stress. R. solanacearum produces a melanin-like brown pigment in the stationary phase when grown in minimal medium containing tyrosine. To gain deeper insight into the genetic determinants involved in melanin production, transposon-inserted mutants of R. solanacearum strain SL341 were screened for strains with defective melanin-producing capability. In addition to one mutant already known to be involved in pyomelanin production (viz., strain SL341D, with disruption of the hydroxphenylpyruvate dioxygenase gene), we identified three other mutants with disruption in the regulatory genes rpoS, hrpG, and oxyR, respectively. Wild-type SL341 produced pyomelanin in minimal medium containing tyrosine whereas the mutant strains did not. Likewise, homogentisate, a major precursor of pyomelanin, was detected in the culture filtrate of the wild-type strain but not in those of the mutant strains. A gene encoding hydroxyphenylpyruvate dioxygenase exhibited a significant high expression in wild type SL341 compared to other mutant strains, suggesting that pyomelanin production is regulated by three different regulatory proteins. However, analysis of the gene encoding homogentisate dioxygenase revealed no significant difference in its relative expression over time in the wild-type SL341 and mutant strains, except for SL341D, at 72 h incubation. The pigmented SL341 strain also exhibited a high tolerance to hydrogen peroxide stress compared with the non-pigmented SL341D strain. Our study suggests that pyomelanin production is controlled by several regulatory factors in R. solanacearum to confer protection under oxidative stress. PMID:27513990

  3. Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

    PubMed

    Elhenawy, Wael; Scott, Nichollas E; Tondo, M Laura; Orellano, Elena G; Foster, Leonard J; Feldman, Mario F

    2016-03-01

    Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex(3), similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum.

  4. Disruption of comA homolog in Ralstonia solanacearum does not impair its twitching motility.

    PubMed

    Barman, Anjan; Buragohain, Chandrika; Ray, Suvendra Kumar

    2017-01-13

    Ralstonia solanacearum is an important phyto-pathogenic bacterium. The bacterium exhibits type IV pili meditated twitching motility that has been implicated in the process of natural transformation in it. A comA gene homolog, alike in several other naturally competent bacteria, has been already reported in this bacterium. However, there are no report of direct link between comA and twitching motility during the natural transformation process in this pathogen. In order to figure out any connection between comA and twitching motility, we created an insertion mutation in comA gene homolog of R. solanacearum F1C1 strain. As anticipated, the insertion mutant (CBRS01 strain) was inefficient for natural transformation. CBRS01 strain was found to be proficient for twitching motility alike the wild-type F1C1. This is interesting since recent findings of Salzer et al. (2016;Environ Microbiol;18:65-74) showed deficiency of twitching motility due to comEC gene (comA homolog) mutation in another naturally competent Gram-negative bacterium Thermus thermophilus. Additionally, we also found CBRS01 strain to be proficient for extracellular cellulase activity and virulence on tomato seedlings. Our findings in this work indicate that an R. solanacearum strain inefficient in undergoing natural transformation can, however, be proficient in exhibiting twitching motility.

  5. Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Shimatani, Mika; Ishikawa, Shiho; Mori, Yuka; Kiba, Akinori; Ohnishi, Kouhei; Tabuchi, Mitsuaki; Hikichi, Yasufumi

    2015-11-02

    Ralstonia solanacearum, a plant pathogenic bacterium causing "bacterial wilt" on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3-hydroxypalmitate (3-OH PAME) was previously identified as the QS signal in strain AW1. However, 3-OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)-3-OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types--(R)-3-OH MAME or (R)-3-OH PAME. These results demonstrate that (R)-3-OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.

  6. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence

    PubMed Central

    2010-01-01

    Background The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats. Results The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole. Conclusions Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic

  7. Diversity of Ralstonia solanacearum in French Guiana expands knowledge of the "emerging ecotype".

    PubMed

    Deberdt, P; Guyot, J; Coranson-Beaudu, R; Launay, J; Noreskal, M; Rivière, P; Vigné, F; Laplace, D; Lebreton, L; Wicker, E

    2014-06-01

    Although bacterial wilt remains a major plant disease throughout South America and the Caribbean, the diversity of prevalent Ralstonia solanacearum populations is largely unknown. The genetic and phenotypic diversity of R. solanacearum strains in French Guiana was assessed using diagnostic polymerase chain reactions and sequence-based (egl and mutS) genotyping on a 239-strain collection sampled on the families Solanaceae and Cucurbitaceae, revealing an unexpectedly high diversity. Strains were distributed within phylotypes I (46.9%), IIA (26.8%), and IIB (26.3%), with one new endoglucanase sequence type (egl ST) found within each group. Phylotype IIB strains consisted mostly (97%) of strains with the emerging ecotype (IIB/sequevar 4NPB). Host range of IIB/4NPB strains from French Guiana matched the original emerging reference strain from Martinique. They were virulent on cucumber; virulent and highly aggressive on tomato, including the resistant reference Hawaii 7996; and only controlled by eggplant SM6 and Surya accessions. The emerging ecotype IIB/4NPB is fully established in French Guiana in both cultivated fields and uncultivated forest, rendering the hypothesis of introduction via ornamental or banana cuttings unlikely. Thus, this ecotype may have originated from the Amazonian region and spread throughout the Caribbean region.

  8. The filamentous phage ϕRSS1 enhances virulence of phytopathogenic Ralstonia solanacearum on tomato.

    PubMed

    Addy, Hardian S; Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2012-03-01

    Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ϕRSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ϕRSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ϕRSS1, resulting in early wilting. Tomato plants inoculated with ϕRSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ϕRSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ϕRSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ϕRSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.

  9. Contrasting recombination patterns and demographic histories of the plant pathogen Ralstonia solanacearum inferred from MLSA

    PubMed Central

    Wicker, Emmanuel; Lefeuvre, Pierre; de Cambiaire, Jean-Charles; Lemaire, Christophe; Poussier, Stéphane; Prior, Philippe

    2012-01-01

    We used multilocus sequence analysis (MLSA) on a worldwide collection of the plant pathogenic Ralstonia solanacearum (Betaproteobacteria) to retrace its complex evolutionary history. Using genetic imprints left during R. solanacearum evolution, we were able to delineate distinct evolutionary complex displaying contrasting dynamics. Among the phylotypes already described (I, IIA, IIB, III, IV), eight groups of strains with distinct evolutionary patterns, named clades, were identified. From our recombination analysis, we identified 21 recombination events that occurred within and across these lineages. Although appearing the most divergent and ancestral phylotype, phylotype IV was inferred as a gene donor for the majority of the recombination events that we detected. Whereas this phylotype apparently fuelled the species diversity, ongoing diversification was mainly detected within phylotype I, IIA and III. These three groups presented a recent expanding population structure, a high level of homologous recombination and evidences of long-distance migrations. Factors such as adaptation to a specific host or intense trading of infected crops may have promoted this diversification. Whether R. solanacearum lineages will eventually evolve in distinct species remains an open question. The intensification of cropping and increase of geographical dispersion may favour situations of phylotype sympatry and promote higher exchange of key factors for host adaptation from their common genetic pool. PMID:22094345

  10. [Advances in studies of the type III secretion system in Ralstonia solanacearum--A review].

    PubMed

    Zhang, Yong; Li, Muyuan; Luo, Feng

    2015-06-04

    Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating plant diseases worldwide. The syringe-like type III secretion system (T3SS) plays a crucial role in its pathogenicity. R. solanacearum uses the T3SS to inject effector proteins (Type III effectors) into the cytoplasm of host cells, causing diseases in susceptible plants or triggering the hypersensitive response in resistant plants. In this article we review recent advances in studies of R. solanacearum T3SS and highlight their unique features.

  11. Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Ling, Ning; Liu, Dongyang; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-11-01

    The volatile organic compounds (VOCs) produced by soil microbes have a significant role in the control of plant diseases and plant growth promotion. In this study, we examined the effect of VOCs produced by Pseudomonas fluorescens strain WR-1 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum. The VOCs produced by P. fluorescens WR-1 exhibited concentration dependent bacteriostatic effect on the growth of R. solanacearum on agar medium and in infested soil. The VOCs of P. fluorescens WR-1 also significantly inhibited the virulence traits of R. solanacearum. The proteomics analysis showed that the VOCs of P. fluorescens WR-1 downregulated cellular proteins of R. solanacearum related to the antioxidant activity, virulence, inclusion body proteins, carbohydrate and amino acid synthesis and metabolism, protein folding and translation, methylation and energy transfer, while the proteins involved in the ABC transporter system, detoxification of aldehydes and ketones, protein folding and translation were upregulated. This study revealed the significance of VOCs of P. fluorescens WR-1 to control the tomato wilt pathogen R. solanacearum. Investigation of the modes of action of biocontrol agents is important to better comprehend the interactions mediated by VOCs in nature to design better control strategies for plant pathogens.

  12. Extract of Syringa oblata: A new biocontrol agent against tobacco bacterial wilt caused by Ralstonia solanacearum.

    PubMed

    Bai, Wanming; Kong, Fanyu; Lin, Yong; Zhang, Chengsheng

    2016-11-01

    Ralstonia solanacearum causes serious wilt disease in tobacco. To effectively control this disease, the antibacterial activity of 95% ethanol extracts from the flower buds of Syringa oblata was examined. Based on GC-MS analysis and an inhibition experiment against R. solanacearum, the main antibacterial component is eugenol. We further determined the effect of eugenol on the physiology, biochemistry, and cellular morphology of R. solanacearum. The results showed that eugenol can destroy wilt bacteria, leading to the disappearance of flagella, the leakage of contents, and the appearance of a cavity. SDS-PAGE showed that eugenol decreased protein content in R. solanacearum, reduced medium carbohydrate utilization, and inhibited CAT and SDH activity. The above results showed that eugenol had a significant inhibitory effect on R. solanacearum and this component has the potential to prevent tobacco bacterial wilt.

  13. Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

    PubMed

    Pensec, Flora; Lebeau, Aurore; Daunay, M C; Chiroleu, Frédéric; Guidot, Alice; Wicker, Emmanuel

    2015-12-01

    For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.

  14. Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum.

    PubMed

    Wang, Keke; Remigi, Philippe; Anisimova, Maria; Lonjon, Fabien; Kars, Ilona; Kajava, Andrey; Li, Chien-Hui; Cheng, Chiu-Ping; Vailleau, Fabienne; Genin, Stéphane; Peeters, Nemo

    2016-05-01

    The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts.

  15. Effect of plant essential oils on Ralstonia solanacearum race 4 causing bacterial wilt of edible ginger

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Palmarosa (Cymbopogon martini), lemongrass (C. citratus) and eucalyptus (Eucalyptus globulus) oils were investigated for their effects on Ralstonia solanacearum race 4, and their potential use as bio-fumigants for treating pathogen- infested edible ginger (Zingiber officinale R.) fields. Three conce...

  16. Functional analysis of Ralstonia solanacearum PrhG regulating the hrp regulon in host plants.

    PubMed

    Zhang, Yong; Chen, Li; Yoshimochi, Takeshi; Kiba, Akinori; Hikichi, Yasufumi; Ohnishi, Kouhei

    2013-08-01

    Genes in the hrp regulon encode component proteins of the type III secretion system and are essential for the pathogenicity of Ralstonia solanacearum. The hrp regulon is controlled by HrpB. We isolated several genes regulating hrpB expression from the Japanese strain OE1-1 using minitransposon mutagenesis. Among them, we mainly focused on two genes, hrpG and prhG, which are the positive regulators of hrpB. Although the global virulence regulator PhcA negatively regulated hrpG expression via prhIR, it positively regulated prhG expression. We further investigated the contrasting regulation of hrpG and prhG by PhcA and speculated that R. solanacearum may switch from HrpG to PrhG for hrpB activation in a cell density-dependent manner. Although the prhG mutant proliferated similarly to the wild-type in leaf intercellular spaces and in xylem vessels of the host plants, it was less virulent than the wild-type. The expression of the popA operon, which belongs to the hrp regulon, was significantly reduced in the prhG mutant by more than half in the leaf intercellular spaces and more than two-thirds in the xylem vessels when compared with the wild-type.

  17. Recent Trends in Control Methods for Bacterial Wilt Diseases Caused by Ralstonia solanacearum

    PubMed Central

    Yuliar; Nion, Yanetri Asi; Toyota, Koki

    2015-01-01

    Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases. PMID:25762345

  18. Detection of Quorum Sensing Molecules and Biofilm Formation in Ralstonia solanacearum.

    PubMed

    Kumar, J Shiva; Umesha, S; Prasad, K Shiva; Niranjana, P

    2016-03-01

    Many bacteria use small diffusible signaling molecules to communicate each other termed as quorum sensing (QS). Most Gram-negative bacteria use acyl homoserine lactone (AHL) as QS signal molecules. Using these signaling molecules, bacteria are able to express specific genes in response to population density. This work aimed to detect the production of QS signal molecules and biofilm formation in Ralstonia solanacearum isolated from various diseased tomato plants with symptoms of bacterial wilt. A total of 30 R. solanacearum strains were investigated for the production of QS signal molecules using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 (pZLR4) biosensor systems. All 30 bacterial isolates from various bacterial wilt-affected tomato plants produced AHL molecules that induced the biosensor. The microtiter plate assay demonstrated that of the 30 bacterial isolates, 60 % formed biofilm, among which four isolates exhibited a higher degree of biofilm formation. The biofilm-inducing factor was purified from these four culture supernatants. The structure of the responsible molecule was solved using nuclear magnetic resonance and mass spectroscopy and was determined to be 2-hydroxy-4-((methylamino)(phenyl)methyl) cyclopentanone (HMCP), which was confirmed by chemical synthesis and NMR. The Confocal laser scanning microscopic analysis showed well-developed biofilm architecture of bacteria when treated with HMCP. The knowledge we obtained from this study will be useful for further researcher on the role of HMCP molecule in biofilm formation.

  19. Recent trends in control methods for bacterial wilt diseases caused by Ralstonia solanacearum.

    PubMed

    Yuliar; Nion, Yanetri Asi; Toyota, Koki

    2015-01-01

    Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases.

  20. Loss of virulence of the phytopathogen Ralstonia solanacearum through infection by φRSM filamentous phages.

    PubMed

    Addy, Hardian S; Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2012-05-01

    φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

  1. Evaluation of Resistance to Ralstonia solanacearum in Tomato Genetic Resources at Seedling Stage.

    PubMed

    Kim, Sang Gyu; Hur, On-Sook; Ro, Na-Young; Ko, Ho-Cheol; Rhee, Ju-Hee; Sung, Jung Sook; Ryu, Kyoung-Yul; Lee, Sok-Young; Baek, Hyung Jin

    2016-02-01

    Bacterial wilt of tomatoes caused by Ralstonia solanacearum is a devastating disease that limits the production of tomato in Korea. The best way to control this disease is using genetically resistant tomato plant. The resistance degree to R. solanacearum was evaluated for 285 tomato accessions conserved in the National Agrobiodiversity Center of Rural Development Administration. These accessions of tomato were originated from 23 countries. Disease severity of tomato accessions was investigated from 7 days to 14 days at an interval of 7 days after inoculation of R. solanacearum under greenhouse conditions. A total of 279 accessions of tomato germplasm were susceptible to R. solanacearum, resulting in wilt and death in 70 to 90% of these plants. Two tomato accessions were moderately resistant to R. solanacearum. Only four accessions showed high resistance against R. solanacearum. No distinct symptom of bacterial wilt appeared on the resistant tomato germplasms for up to 14 days after inoculation of R. solanacearum. Microscopy of resistant tomato stems infected with R. solanacearum revealed limited bacterial spread with thickening of pit membrane and gum production. Therefore, these four resistant tomato germplasms could be used in tomato breeding program against bacterial wilt.

  2. Nitrate assimilation contributes to Ralstonia solanacearum root attachment, stem colonization, and virulence.

    PubMed

    Dalsing, Beth L; Allen, Caitilyn

    2014-03-01

    Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium's gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum's sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.

  3. Multihost experimental evolution of the pathogen Ralstonia solanacearum unveils genes involved in adaptation to plants.

    PubMed

    Guidot, Alice; Jiang, Wei; Ferdy, Jean-Baptiste; Thébaud, Christophe; Barberis, Patrick; Gouzy, Jérôme; Genin, Stéphane

    2014-11-01

    Ralstonia solanacearum, the causal agent of a lethal bacterial wilt plant disease, infects an unusually wide range of hosts. These hosts can further be split into plants where R. solanacearum is known to cause disease (original hosts) and those where this bacterium can grow asymptomatically (distant hosts). Moreover, this pathogen is able to adapt to many plants as supported by field observations reporting emergence of strains with enlarged pathogenic properties. To investigate the genetic bases of host adaptation, we conducted evolution experiments by serial passages of a single clone of the pathogen on three original and two distant hosts over 300 bacterial generations and then analyzed the whole-genome of nine evolved clones. Phenotypic analysis of the evolved clones showed that the pathogen can increase its fitness on both original and distant hosts although the magnitude of fitness increase was greater on distant hosts. Only few genomic modifications were detected in evolved clones compared with the ancestor but parallel evolutionary changes in two genes were observed in independent evolved populations. Independent mutations in the regulatory gene efpR were selected for in three populations evolved on beans, a distant host. Reverse genetic approaches confirmed that these mutations were associated with fitness gain on bean plants. This work provides a first step toward understanding the within-host evolutionary dynamics of R. solanacearum during infection and identifying bacterial genes subjected to in planta selection. The discovery of EfpR as a determinant conditioning host adaptation of the pathogen illustrates how experimental evolution coupled with whole-genome sequencing is a potent tool to identify novel molecular players involved in central life-history traits.

  4. Isolation and screening of phlD (+) plant growth promoting rhizobacteria antagonistic to Ralstonia solanacearum.

    PubMed

    Ramadasappa, Srinivasamurthy; Rai, Ashwani K; Jaat, Ranjeet Singh; Singh, Aqbal; Rai, Rhitu

    2012-04-01

    Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD (+) bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD (+) antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92-5.33%), 25 produced indole acetic acid (1.63-7.78 μg ml(-1)) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD (+)) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD (+) bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD (+)) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant(-1) respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD (+) isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.

  5. TssB is essential for virulence and required for type VI secretion system in Ralstonia solanacearum.

    PubMed

    Zhang, Liqing; Xu, Jingsheng; Xu, Jin; Zhang, Hao; He, Liyuan; Feng, Jie

    2014-09-01

    The type VI secretion system (T6SS) is recently discovered machinery in Gram-negative bacteria for translocation of proteins and also is required for full virulence. TssB is a highly conserved protein among the T6SSs, and indispensable for composition and function of T6S. The plant pathogenic bacterium Ralstonia solanacearum also harbours T6SS gene clusters, and a homologue of TssB, hereafter designated as TssBRS, but up to date its characterization and function remain unclear. In this study, we showed that TssBRS of R. solanacearum was required for secretion of Hcp, the haemolysin coregulated protein and a hallmark of T6S pathway. Deletion of tssBRS in R. solanacearum GMI1000 strain resulted in defect of biofilm formation, and the expression of the flagella operon is decreased, leading to decreased motility. More importantly, tssBRS mutant strain had significantly attenuated its virulence on tomato plants. TssB is essential for virulence and required for type VI secretion system in R. solanacearum.

  6. Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2.

    PubMed

    Meng, Fanhong; Babujee, Lavanya; Jacobs, Jonathan M; Allen, Caitilyn

    2015-01-01

    While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.

  7. Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2

    PubMed Central

    Meng, Fanhong; Babujee, Lavanya; Jacobs, Jonathan M.; Allen, Caitilyn

    2015-01-01

    While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2. PMID:26445498

  8. Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco

    PubMed Central

    Lowe-Power, Tiffany M.; Jacobs, Jonathan M.; Ailloud, Florent; Fochs, Brianna; Prior, Philippe

    2016-01-01

    ABSTRACT Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum. SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. PMID:27329752

  9. An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum.

    PubMed Central

    Huang, Q; Allen, C

    1997-01-01

    Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors. These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence. The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units. The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level. PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB. The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively. The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method. However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all. These results suggest that PehB is required for a wild-type level of virulence in R. solanacearum although its individual role in wilt disease development may be minor. Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R. solanacearum. PMID:9393701

  10. Antibacterial activity of the flavonoids from Dalbergia odorifera on Ralstonia solanacearum.

    PubMed

    Zhao, Xiabo; Mei, Wenli; Gong, Mingfu; Zuo, Wenjian; Bai, Hongjin; Dai, Haofu

    2011-11-25

    Phytohemical investigation on the heartwood of Dalbergia odorifera resulted in the isolation of nine flavonoids. Their structures were elucidated as sativanone (1), (3R)-vestitone (2), (3R)-2',3',7-trihydroxy-4'-methoxyisoflavanone (3), (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone (4), carthamidin (5), liquiritigenin (6), isoliquiritigenin (7), (3R)-vestitol (8), and sulfuretin (9) based on their spectral data. All compounds were evaluated for their inhibitory activity against Ralstonia solanacearum. This is the first report about anti-R. solanacearum activity of the compounds from D. odorifera.

  11. Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

    PubMed Central

    Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

    2012-01-01

    Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

  12. Only one of the five Ralstonia solanacearum long-chain 3-ketoacyl-acyl carrier protein synthase homologues functions in fatty acid synthesis.

    PubMed

    Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E; Wang, Haihong

    2012-03-01

    Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C(16:0)), palmitoleic (C(16:1)) and cis-vaccenic (C(18:1)) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I.

  13. Necessity of OxyR for the Hydrogen Peroxide Stress Response and Full Virulence in Ralstonia solanacearum ▿ †

    PubMed Central

    Flores-Cruz, Zomary; Allen, Caitilyn

    2011-01-01

    The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence. PMID:21803891

  14. Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum.

    PubMed

    Flores-Cruz, Zomary; Allen, Caitilyn

    2011-09-01

    The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.

  15. Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum.

    PubMed

    Kawasaki, Takeru; Narulita, Erlia; Matsunami, Minaho; Ishikawa, Hiroki; Shimizu, Mio; Fujie, Makoto; Bhunchoth, Anjana; Phironrit, Namthip; Chatchawankanphanich, Orawan; Yamada, Takashi

    2016-05-01

    The genome organization, gene structure, and host range of five podoviruses that infect Ralstonia solanacearum, the causative agent of bacterial wilt disease were characterized. The phages fell into two distinctive groups based on the genome position of the RNA polymerase gene (i.e., T7-type and ϕKMV-type). One-step growth experiments revealed that ϕRSB2 (a T7-like phage) lysed host cells more efficiently with a shorter infection cycle (ca. 60 min corresponding to half the doubling time of the host) than ϕKMV-like phages such as ϕRSB1 (with an infection cycle of ca. 180 min). Co-infection experiments with ϕRSB1 and ϕRSB2 showed that ϕRSB2 always predominated in the phage progeny independent of host strains. Most phages had wide host-ranges and the phage particles usually did not attach to the resistant strains; when occasionally some did, the phage genome was injected into the resistant strain's cytoplasm, as revealed by fluorescence microscopy with SYBR Gold-labeled phage particles.

  16. prhKLM genes of Ralstonia solanacearum encode novel activators of hrp regulon and are required for pathogenesis in tomato.

    PubMed

    Zhang, Yong; Kiba, Akinori; Hikichi, Yasufumi; Ohnishi, Kouhei

    2011-04-01

    The genes in the hrp regulon encode the proteins composing type III secretion system in Ralstonia solanacearum. The hrp regulon is positively controlled by HrpB, and hrpB expression is activated by both HrpG and PrhG. We have identified three genes, prhK, prhL, and prhM, which positively control the hrp regulon in strain OE1-1. These genes are likely to form an operon, and this operon is well conserved in the genera Ralstonia and Burkholderia. This indicates that the operon is not specific to the plant pathogens. Mutations in each of these three genes abolished hrpB and prhG expression. prhK, prhL, and prhM mutant strains lost pathogenicity toward tomato completely, and they were less virulent toward tobacco. PrhK and PrhL share sequence similarity with allophanate hydrolase and PrhM with LamB. This suggests that the three gene products are not transcriptional regulators in the strict sense, but regulate hrp regulon indirectly. This novel class of virulence-related genes will mark the beginning of new findings regarding the overall infection mode of R. solanacearum.

  17. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues

    PubMed Central

    Spraker, Joseph E; Sanchez, Laura M; Lowe, Tiffany M; Dorrestein, Pieter C; Keller, Nancy P

    2016-01-01

    Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus. PMID:26943626

  18. Transcriptome analysis of quantitative resistance-specific response upon Ralstonia solanacearum infection in tomato.

    PubMed

    Ishihara, Takeaki; Mitsuhara, Ichiro; Takahashi, Hideki; Nakaho, Kazuhiro

    2012-01-01

    Bacterial wilt, caused by the soil-borne bacterium Ralstonia solanacearum, is a lethal disease of tomato, but the molecular mechanisms of the host resistance responses to R. solanacearum remain unclear. In this study, we report the first work describing the transcriptome of cultivar resistance and susceptible tomato cultivar after inoculation with R. solanacearum. To elucidate the characteristics of resistance early in the interaction, we analyzed microarrays for resistant cultivar LS-89 and susceptible cultivar Ponderosa 1 day after stem inoculation. No change in gene expression was detected for Ponderosa, but expression levels of over 140 genes, including pathogenesis-related, hormone signaling and lignin biosynthesis genes, increased in LS-89. Expression of β-1,3-glucanase genes increased substantially. In an immunohistochemical study, glucanase in LS-89 accumulated in the xylem and pith tissues surrounding xylem vessels filled with R. solanacearum. The expression of these genes also increased in four other resistant cultivars, but changed little in four susceptible cultivars in response to R. solanacearum, suggesting that similar reactions occur in other cultivars. These gene expression profiles will serve as fundamental information to elucidate the molecular mechanisms in the resistance response to R. solanacearum in tomato.

  19. A MotN Mutant of Ralstonia solanacearum Is Hypermotile and Has Reduced Virulence ▿ †

    PubMed Central

    Meng, Fanhong; Yao, Jian; Allen, Caitilyn

    2011-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that causes bacterial wilt disease on many plant species. We previously showed that swimming motility contributes to virulence of this bacterium in the early stages of host invasion and colonization. In this study we identified a new negative regulator of motility, named motN, that is located in a cluster of motility-related genes. A motN mutant was hypermotile both on 0.3% agar motility plates and in rich and minimal medium broth. However, like its wild-type parent, it was largely nonmotile inside plants. The motN mutant cells appeared hyperflagellated, and sheared cell protein preparations from motN contained more flagellin than preparations from wild-type cells. The motN strain was significantly reduced in virulence in a naturalistic soil soak assay on tomato plants. However, the motN mutant had wild-type virulence when it was inoculated directly into the plant vascular system. This suggests that motN makes its contribution to virulence early in disease development. The motN mutant formed weaker biofilms than the wild type, but it attached normally to tomato roots and colonized tomato stems as well as its wild-type parent. Phenotypic analysis and gene expression studies indicated that MotN directly or indirectly represses transcription of the major motility regulator FlhDC. MotN was also connected with other known motility and virulence regulators, PehSR, VsrBC, and VsrAD, via uncertain mechanisms. Together, these results demonstrate the importance of precise regulation of flagellum-mediated motility in R. solanacearum. PMID:21421761

  20. Development of the sensitive lateral flow immunoassay with silver enhancement for the detection of Ralstonia solanacearum in potato tubers.

    PubMed

    Panferov, Vasily G; Safenkova, Irina V; Varitsev, Yury A; Drenova, Natalia V; Kornev, Konstantin P; Zherdev, Anatoly V; Dzantiev, Boris B

    2016-05-15

    Ralstonia solanacearum is a dangerous and economically important pathogen of potatoes and other agricultural crops. Therefore, rapid and sensitive methods for its routine diagnostics are necessary. The aim of this study was to develop a rapid control method for R. solanacearum with a low limit of detection (LOD) based on a lateral flow immunoassay (LFIA) with silver enhancement. To minimize the LOD, the membrane type, antibody amount for conjugation with gold nanoparticles, conjugate concentration and antibody concentration in the analytical zone were optimized. Silver enhancement was used to decrease the LOD of the LFIA. For silver enhancement, release fiberglass membranes with pre-absorbed silver lactate and hydroquinone were placed on the analytical zone, and a drop of silver lactate was added. The LFIA with silver enhancement was found to be 10-fold more sensitive (LOD 2×10(2) CFU/mL; 20 min) in comparison with the common analysis (LOD 2×10(3) CFU/mL; 10 min). The specificity of the developed LFIA was studied using different strains of R. solanacearum (54 samples) and other widespread bacterial pathogens (18 samples). The LFIA detected all tested strains, whereas non-specific reactions were not observed. The developed tests were used for the control of bacteria in extracts of infected and non-infected potato tubers, and the quantitative analysis results (based on the densitometry of line colouration) were confirmed by ELISA with a correlation coefficient equal to 0.965.

  1. Evaluation of antibacterial effects of carbon nanomaterials against copper-resistant Ralstonia solanacearum.

    PubMed

    Wang, Xiuping; Liu, Xueqin; Han, Heyou

    2013-03-01

    In this paper, we investigated the antibacterial activity and the action mode of carbon nanomaterials (CNMs) against the copper-resistant plant pathogenic bacterium Ralstonia solanacearum (R. solanacearum). Single-walled carbon nanotubes (SWCNTs) dispersion was found to show the strongest antibacterial activity, sequentially followed by graphene oxide (GO), multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (rGO) and fullerene (C(60)). Our investigation of the antibacterial mechanism of SWCNTs and GO indicated that the damage to the cell membrane leads to the release of cytoplasm materials from the bacterium, which is the causative factor for the inactivation of R. solanacearum bacterial cells. The superior antibacterial effect, and the novel antibacterial mode of SWCNTs and GO suggest that those carbon nanomaterials may have important applications in the control of plant bacterial diseases.

  2. Effects of compost addition and simulated solarisation on the fate of Ralstonia solanacearum biovar 2 and indigenous bacteria in soil.

    PubMed

    Schönfeld, J; Gelsomino, A; Overbeek, L S; Gorissen, A; Smalla, K; Elsas, J D

    2003-02-01

    Abstract The effects of compost addition and simulated solarisation of soil on the survival of Ralstonia solanacearum biovar 2 strain 1609, as well as on the structure of indigenous soil bacterial communities, were analysed. In addition, effects on the invasion of susceptible test plants by strain 1609 were assessed. In untreated soil in microcosms and the field, strain 1609 showed slow progressive declines, from 10(6)-10(7) to roughly 10(4)-10(5) CFU per g dry soil in around 60 days. When these soils were used in suppressiveness tests, a majority of plants developed symptoms of wilting and revealed the presence of the pathogen in their lower stem parts, as evidenced by immunofluorescence colony staining (IFC) and polymerase chain reaction (PCR). Solarisation of unamended soil did not drastically affect R. solanacearum survival or plant invasiveness. However, the addition of household compost resulted in enhanced R. solanacearum population decline rates, as well as reduced numbers of diseased plants in suppressiveness tests. Combined solarisation and compost addition yielded differential results between microcosms and the field. Some healthy-looking plants, primarily from soils treated with compost, revealed the latent presence of strain 1609 in the lower stem parts. The eubacterial and beta-subgroup proteobacterial communities in the differentially treated soil microcosms were rather stable, as evidenced by analysis of PCR-denaturing gradient gel electrophoresis (DGGE) generated molecular profiles. However, compost amendment clearly induced changes in these communities, which were detectable until the end of the experiment; two major bands, affiliated with Variovorax paradoxus and Aquaspirillum psychrophylum, were associated with the compost amendment. The decrease in abundance of R. solanacearum in the compost-amended soils was confirmed by the DGGE profiles.

  3. Preventing Ralstonia solanacearum adhesion with glycans from cashew, cocoa, coffee, pumpkin, and tomato seed extract.

    PubMed

    Rachmaninov, Ofra; Zinger-Yosovich, Keren D; Gilboa-Garber, Nechama

    2012-07-01

    Ralstonia solanacearum wilts many plants, causing heavy agricultural losses. Its pathogenic strain ATCC 11696 produces 2 hemagglutinating lectins: RSL and RS-IIL. These lectins may bind to terminal l-fucose-, d-arabinose-, and d-mannose-bearing seedling xylem cell wall glycans, thus enabling pathogen adhesion to them, with devastating infection establishment. Blocking the active sites of these lectins with seed embryo-surrounding oligo- and poly-saccharides hampers binding of the lectins to the embryos. The current study shows that seeds of cashew, cocoa, coffee, pumpkin, and tomato contain low and high molecular mass glycans that block RSL and RS-IIL (like its homologous Pseudomonas aeruginosa PA-IIL lectin). The blocking of the pathogen lectins, which is attributable to the documented composition of the oligo- and poly-saccharides of these seeds, is similar to that observed with animal glycoproteins of avian egg whites (protecting their embryos from infections) and of milk and royal jelly, which likewise protect mammal and bee neonates, respectively. RSL was most strongly inhibited by cashew seed glycans, and RS-IIL by coffee seed glycans. Western blot analyses with these lectins instead of antibodies revealed the hitherto undescribed presence of lectin-binding glycoproteins in the coffee, pumpkin, tomato, and cashew (but not cocoa) seeds. The use of these lectins for unveiling potent embryo-protecting seed glycans might be helpful for seedling-bioprotection projects similar to those planned for animal protection against antibiotic-resistant infections.

  4. New Insights into the Antibacterial Activity of Hydroxycoumarins against Ralstonia solanacearum.

    PubMed

    Yang, Liang; Ding, Wei; Xu, Yuquan; Wu, Dousheng; Li, Shili; Chen, Juanni; Guo, Bing

    2016-04-08

    Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM) and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt.

  5. Potential Interactions between Salmonella enterica and Ralstonia solanacearum in tomato plants.

    PubMed

    Pollard, Stephanie; Barak, Jeri; Boyer, Renee; Reiter, Mark; Gu, Ganyu; Rideout, Steven

    2014-02-01

    Over the past decade, the Eastern Shore of Virginia (ESV) has been implicated in at least four outbreaks of salmonellosis associated with tomato, all originating from the same serovar, Salmonella enterica serovar Newport. In addition to Salmonella Newport contamination, the devastating plant disease bacterial wilt, caused by the phytopathogen Ralstonia solanacearum, threatens the sustainability of ESV tomato production. Bacterial wilt is present in most ESV tomato fields and causes devastating yield losses each year. Although the connection between bacterial wilt and tomato-related salmonellosis outbreaks in ESV is of interest, the relationship between the two pathogens has never been investigated. In this study, tomato plants were root dip inoculated with one of four treatments: (i) 8 log CFU of Salmonella Newport per ml, (ii) 5 log CFU of R. solanacearum per ml, (iii) a coinoculation of 8 log CFU of Salmonella Newport per ml plus 5 log CFU of R. solanacearum per ml, and (iv) sterile water as control. Leaf, stem, and fruit samples were collected at the early-green-fruit stage, and S. enterica contamination in the internal tissues was detected. S. enterica was recovered in 1.4 and 2.9% of leaf samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. S. enterica was recovered from 1.7 and 3.5% of fruit samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. There were significantly more stem samples from plants coinoculated with Salmonella Newport plus R. solanacearum that were positive for S. enterica (18.6%) than stem samples collected from plants inoculated with Salmonella Newport only (5.7%). Results suggested that R. solanacearum could influence S. enterica survival and transportation throughout the internal tissues of tomato plants.

  6. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Wang, Jichen; Wu, Yuncheng; Ling, Ning; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-09-01

    The production of volatile organic compounds (VOCs) by microbes is an important characteristic for their selection as biocontrol agents against plant pathogens. In this study, we identified the VOCs produced by the biocontrol strain Bacillus amyloliquefaciens T-5 and evaluated their impact on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum. The results showed that the VOCs of strain T-5 significantly inhibited the growth of R. solanacearum in agar medium and in soil. In addition, VOCs significantly inhibited the motility traits, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides by R. solanacearum. However, no effect of VOCs on the production of hydrolytic enzymes by R. solanacearum was observed. The strain T-5 produced VOCs, including benzenes, ketones, aldehydes, alkanes, acids, and one furan and naphthalene compound; among those, 13 VOCs showed 1-10 % antibacterial activity against R. solanacearum in their produced amounts by T-5; however, the consortium of all VOCs produced on agar medium, in sterilized soil, and in natural soil showed 75, 62, and 85 % growth inhibition of R. solanacearum, respectively. The real-time PCR analysis further confirmed the results when the expression of different virulence- and metabolism-related genes in R. solanacearum cells was decreased after exposure to the VOCs of strain T-5. The results of this study clearly revealed the significance of VOCs in the control of plant pathogens. This information would help to better comprehend the microbial interactions mediated by VOCs in nature and to develop safer strategies to control plant disease.

  7. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    PubMed Central

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-01-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  8. Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum

    PubMed Central

    Wu, Dousheng; Ding, Wei; Zhang, Yong; Liu, Xuejiao; Yang, Liang

    2015-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway. PMID:26732647

  9. TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity

    PubMed Central

    Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

    2016-01-01

    Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47–91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other’s EBEs. Investigation of sequence divergence

  10. Detection of Ralstonia solanacearum from asymptomatic tomato plants, irrigation water, and soil through non-selective enrichment medium with hrp gene-based bio-PCR.

    PubMed

    Singh, Dinesh; Sinha, Shweta; Yadav, D K; Chaudhary, Garima

    2014-08-01

    Bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) Yabuuchi et al. (Microbiol Immunol 39:897-904, 1995) is a serious disease, which causes losses up to 60 % depending on environmental conditions, soil property, and cultivars. In present investigation, nucleotide sequences of virulence, hypersensitive response and pathogenicity (hrp) gene were used to design a pair of primer (Hrp_rs 2F: 5'-AGAGGTCGACGCGATACAGT-3' and Hrp_rs 2R: 5'-CATGAGCAAGGACGAAGTCA-3') for amplification of bacterial genome. The genomic DNA of 27 isolates of R. solanacearum race 1 biovar 3 & 4 was amplified at 323 bp. The specificity of primer was tested on 13 strains of R. solanacearum with other group of bacteria such as Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, and X. citri subsp. citri. Primer amplified DNA fragment of R. solanacearum at 323 bp. The sensitivity of the primer was 200 cfu/ml and improved further detection level by using non-specific enrichment medium casamino acids-pepton-glucose broth followed by PCR (BIO-PCR). Out of 130 samples of asymptomatic tomato plants, irrigation water, and soil collected from bacterial wilt infested field in different agro-climatic regions of India, R. solanacearum was detected from 86.9, 88.5, and 90.9 per cents samples using BIO-PCR, respectively. The primer was found specific for detecting viable and virulent strains of R. solanacearum and useful for the diagnosis of R. solanacearum in tomato seedlings and monitoring of pathogen in irrigation water and soil.

  11. Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species.

    PubMed

    Um, Hae Young; Chung, Eunsook; Lee, Jai-Heon; Lee, Seon-Woo

    2011-04-01

    Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.

  12. Ralstonia solanacearum RSp0194 Encodes a Novel 3-Keto-Acyl Carrier Protein Synthase III.

    PubMed

    Mao, Ya-Hui; Ma, Jin-Cheng; Li, Feng; Hu, Zhe; Wang, Hai-Hong

    2015-01-01

    Fatty acid synthesis (FAS), a primary metabolic pathway, is essential for survival of bacteria. Ralstonia solanacearum, a β-proteobacteria member, causes a bacterial wilt affecting more than 200 plant species, including many economically important plants. However, thus far, the fatty acid biosynthesis pathway of R. solanacearum has not been well studied. In this study, we characterized two forms of 3-keto-ACP synthase III, RsFabH and RsFabW, in R. solanacearum. RsFabH, the homologue of Escherichia coli FabH, encoded by the chromosomal RSc1050 gene, catalyzes the condensation of acetyl-CoA with malonyl-ACP in the initiation steps of fatty acid biosynthesis in vitro. The RsfabH mutant lost de novo fatty acid synthetic ability, and grows in medium containing free fatty acids. RsFabW, a homologue of Pseudomonas aeruginosa PA3286, encoded by a megaplasmid gene, RSp0194, condenses acyl-CoA (C2-CoA to C10-CoA) with malonyl-ACP to produce 3-keto-acyl-ACP in vitro. Although the RsfabW mutant was viable, RsfabW was responsible for RsfabH mutant growth on medium containing free fatty acids. Our results also showed that RsFabW could condense acyl-ACP (C4-ACP to C8-ACP) with malonyl-ACP, to produce 3-keto-acyl-ACP in vitro, which implies that RsFabW plays a special role in fatty acid synthesis of R. solanacearum. All of these data confirm that R. solanacearum not only utilizes acetyl-CoA, but also, utilizes medium-chain acyl-CoAs or acyl-ACPs as primers to initiate fatty acid synthesis.

  13. The in planta transcriptome of Ralstonia solanacearum: conserved physiological and virulence strategies during bacterial wilt of tomato.

    PubMed

    Jacobs, Jonathan M; Babujee, Lavanya; Meng, Fanhong; Milling, Annett; Allen, Caitilyn

    2012-01-01

    Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 10(8) to 10(9) CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 10(8) CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. IMPORTANCE Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical

  14. Genetic diversity of the bacterial wilt pathogen Ralstonia solanacearum using a RAPD marker.

    PubMed

    Nishat, Sayeda; Hamim, Islam; Khalil, M Ibrahim; Ali, Md Ayub; Hossain, Muhammed Ali; Meah, M Bahadur; Islam, Md Rashidul

    2015-11-01

    Bacterial wilt caused by Ralstonia solanacearum is a destructive disease of many economically important crop species. A significant variation in wilt incidence and severity in eggplant and potato was observed among the growing areas surveyed. R. solanacearum isolates obtained both from eggplant and potato belong to biovar III, while isolates from eggplant belong to race 1 and isolates obtained from potato belong to race 3. Random amplified polymorphic DNA (RAPD) technique was used as a tool for assessing genetic variation and relationship among seven isolate groups of R. solanacearum viz., RsB-1, RsB-2, RsB-3, RsP-1, RsP-2, RsP-3 and RsP-4, consisting in a total of 28 isolates. Out of the RAPD markers used, amplification with four decamer primers produced 70 bands with sizes ranging from 100 to 1400 bp. Out of 70 bands, 68 bands (97.06%) were polymorphic and two bands (2.94%) were monomorphic amongst the seven R. solanacearum isolates group. The Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram constructed from Nei's genetic distance produced two main clusters of the seven isolates of R. solanacearum. The isolates RsB-1, RsB-2, RsB-3 and R-4 grouped in cluster І, while RsP-2, RsP-3 and RsP-4 grouped in cluster ІІ. The highest intra-variety similarity index (Si) was found in RsB-1 isolate (86.35%) and the lowest one in RsP-2 (56.59%). The results indicated that relatively higher and lower levels of genetic variation were found in RsP-3 and RsB-3, respectively. The coefficient of gene differentiation (G(st)) was 0.5487, reflecting the existence of a high level of genetic variations among seven isolates of R. solanacearum. Comparatively higher genetic distance (0.4293) and lower genetic identity (0.6510) were observed between RsB-2 and RsP-4 combinations. The lowest genetic distance (0.0357) and highest genetic identity (0.9650) were found in RsB-1 vs. RsB-2 pair. Thus, RAPD offers a potentially simple, rapid and reliable method to evaluate

  15. Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

    PubMed

    de Lange, Orlando; Schreiber, Tom; Schandry, Niklas; Radeck, Jara; Braun, Karl Heinz; Koszinowski, Julia; Heuer, Holger; Strauß, Annett; Lahaye, Thomas

    2013-08-01

    Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.

  16. Elicitor-Induced Defense Responses in Solanum lycopersicum against Ralstonia solanacearum

    PubMed Central

    Kar, Itishree; Mukherjee, Arup K.; Acharya, Priyambada

    2013-01-01

    We investigated on important parameters of induced resistance in hydroponic tomato (Solanum lycopersicum) against Ralstonia solanacearum using the elicitors chitosan (CHT), salicylic acid (SA), and jasmonic acid (JA). The increase in total phenolic content of roots by the elicitors was significantly higher than control. Most pronounced increase in lignin synthesis was triggered by SA followed by CHT. At 24 h post-elicitation (hpe), the activity of phenylalanine ammonia lyase was 4.5 times higher than control elicited by CHT. The peroxidase activity was about 86 nkat/mg protein at 24 hpe in case of SA and 78 nkat/mg protein in case of CHT. The activity of polyphenol oxidase increased several folds by the elicitors. Cinnamyl alcohol dehydrogenase activity increased to the maximum at 48 hpe under the influence of CHT. The results indicate that the elicitors SA and CHT induced effective defense responses in tomato plants against R. solanacearum. This was evident from reduced vascular browning and wilting symptoms of tomato plants treated with SA and CHT and challenged subsequently with R. solanacearum. This reduced disease incidence in tomato by SA and CHT may be a result of cell wall strengthening through deposition of lignin and the coincident induction of defense enzymes. PMID:24187521

  17. Comparative behavior of Ralstonia solanacearum biovar 2 in diverse plant species.

    PubMed

    Alvarez, B; Vasse, J; Le-Courtois, V; Trigalet-Démery, D; López, M M; Trigalet, A

    2008-01-01

    Ralstonia solanacearum causes bacterial wilt in numerous plant species worldwide. Although biovar 2 mostly affects solanaceous crops, identification of new hosts remains a matter of concern since there is still no clear-cut distinction between host and nonhost plants. In this work we provide data based on histological studies on the status of 20 plant species, most of them of potential interest in crop rotation. Plants were watered with a beta-glucuronidase-expressing derivative of R. solanacearum biovar 2, and after a month of incubation, sections of roots and stems were analyzed to localize the pathogen on surface, in cortex and/or xylem. Depending on whether the xylem was colonized or not, plants were classified as hosts or nonhosts, respectively. Hosts generally affected in a few xylem vessels or occasionally in all xylem bundles were classified as tolerant. These included some cabbage, kidney bean, and rutabaga cultivars, and the weed bittersweet nightshade (Solanum dulcamara). Nonhosts were the cultivars tested of alfalfa, barley, black radish, carrot, celery, colocynth, fennel, fiber flax, field bean, field pea, horseradish, maize, and zucchini. However, barley and maize, though nonhosts, may act as reservoirs for the pathogen. The present work constitutes a basis for further studies on cropping systems in fields where R. solanacearum has been detected.

  18. Antibacterial activity of Lansiumamide B to tobacco bacterial wilt (Ralstonia solanacearum).

    PubMed

    Li, Lichun; Feng, Xiujie; Tang, Ming; Hao, Wenbo; Han, Yun; Zhang, Guobin; Wan, Shuqing

    2014-01-01

    Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases of tobacco in the area of tobacco cultivation. As there is no effective control method for tobacco bacterial wilt diseases, developing new antibacterial agents in tobacco will make great practical sense. The antibacterial activity against R. solanacearum of Lansiumamide B which is isolated from the seeds of Clausena lansium is reported in this paper for the first time. The bioassay results indicate that Lansiumamide B could completely inhibit the growth of R. solanacearum at the concentration of 125 mg/L in vitro, the EC50 and EC90 are 48.82 mg/L and 86.26 mg/L, respectively. The result of pot experiments indicates that the control efficiency of the Lansiumamide B on tobacco bacterial wilt are 95.84%, 91.67% and 86.38% at 7 days, 14 days and 21 days after treatment at the concentration of 100mg/kg, respectively, nearly 40 times higher than Streptomycin, a special fungicide to the disease, at 21 days after treatment with root irrigation method. These results suggest that Lansiumamide B has the potential of developing as a new type of plant-type fungicide on controlling the diseases of tobacco bacterial wilt.

  19. Heat shock, with recovery, promotes protection of Nicotiana tabacum during subsequent exposure to Ralstonia solanacearum.

    PubMed

    Byth-Illing, Heather-Anne; Bornman, Liza

    2014-03-01

    Host-pathogen interactions in plants are complex and potentially influenced by heat shock/stress (HS). Host HS proteins (HSPs) induced prior to bacterial exposure may facilitate the folding of newly synthesized defense proteins and promote incompatible host-pathogen interactions. We hypothesized that a non-lethal HS, with recovery, promotes protection of Nicotiana tabacum during subsequent exposure to avirulent soilborne necrotrophic pathogen Ralstonia solanacearum. The objective of this study included investigating the effects of HS with or without recovery on the outcome of bacterial exposure to a virulent and avirulent biovar of R. solanacearum in N. tabacum cell suspensions. This was assessed by quantifying host Hsp70/Hsc70 levels, mitochondrial electron (e (-)) transport activity as a marker of viability, and phosphatidylserine externalization and DNA fragmentation as markers of apoptosis. Our findings support the hypothesis that HS, with recovery, promotes protection of N. tabacum during subsequent exposure to R. solanacearum, suggesting a role for Hsp70/Hsc70 in the observed protection of e (-) transport, increased apoptosis, and DNA fragmentation.

  20. Ralstonia solanacearum Differentially Colonizes Roots of Resistant and Susceptible Tomato Plants.

    PubMed

    Caldwell, Denise; Kim, Bong-Suk; Iyer-Pascuzzi, Anjali S

    2017-03-21

    Ralstonia solanacearum is the causal agent of bacterial wilt and infects over 200 plant species in 50 families. The soilborne bacterium is lethal to many solanaceous species, including tomato. Although resistant plants can carry high pathogen loads (between 10(5) and 10(8) CFU/g fresh weight), the disease is best controlled by the use of resistant cultivars, particularly resistant rootstocks. How these plants have latent infections yet maintain resistance is not clear. R. solanacearum first infects the plant through the root system and, thus, early root colonization events may be key to understanding resistance. We hypothesized that the distribution and timing of bacterial invasion differed in roots of resistant and susceptible tomato cultivars. Here, we use a combination of scanning electron microscopy and light microscopy to investigate R. solanacearum colonization in roots of soil-grown resistant and susceptible tomato cultivars at multiple time points after inoculation. Our results show that colonization of the root vascular cylinder is delayed in resistant 'Hawaii7996' and that, once bacteria enter the root vascular tissues, colonization in the vasculature is spatially restricted. Our data suggest that resistance is due, in part, to the ability of the resistant cultivar to restrict bacterial root colonization in space and time.

  1. Hydroxycinnamic acid degradation, a broadly conserved trait, protects Ralstonia solanacearum from chemical plant defenses and contributes to root colonization and virulence

    PubMed Central

    Lowe, Tiffany M.; Ailloud, Florent; Allen, Caitilyn

    2014-01-01

    Plants produce hydroxycinnamic acid defense compounds (HCAs) to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a Δfcs (feruloyl-CoA synthetase) mutant that cannot degrade HCAs was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen (1) grow, as a carbon source; (2) spread, by reducing physical barriers HCA-derived; and (3) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCAs in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCAs are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCAs: caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCAs contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity. PMID:25423265

  2. Hydroxycinnamic Acid Degradation, a Broadly Conserved Trait, Protects Ralstonia solanacearum from Chemical Plant Defenses and Contributes to Root Colonization and Virulence.

    PubMed

    Lowe, Tiffany M; Ailloud, Florent; Allen, Caitilyn

    2015-03-01

    Plants produce hydroxycinnamic acid (HCA) defense compounds to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase (Δfcs) mutant that cannot degrade HCA was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCA are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCA, namely, caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCA contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity.

  3. Silicon-mediated tomato resistance against Ralstonia solanacearum is associated with modification of soil microbial community structure and activity.

    PubMed

    Wang, Lei; Cai, Kunzheng; Chen, Yuting; Wang, Guoping

    2013-05-01

    Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease of Solanaceae crops. In this study, the soil microbial effects of silicon-induced tomato resistance against R. solanacearum were investigated through pot experiment. The results showed that exogenous 2.0 mM Si treatment reduced the disease index of bacterial wilt by 19.18 % to 52.7 % compared with non-Si-treated plants. The uptake of Si was significantly increased in the Si-treated tomato plants, where the Si content was higher in the roots than that in the shoots. R. solanacearum inoculation resulted in a significant increase of soil urease activity and reduction of soil sucrase activity, but had no effects on soil acid phosphatase activity. Si supply significantly increased soil urease and soil acid phosphatase activity under pathogen-inoculated conditions. Compared with the non-inoculated treatment, R. solanacearum infection significantly reduced the amount of soil bacteria and actinomycetes by 52.5 % and 16.5 %, respectively, but increased the ratio of soil fungi/soil bacteria by 93.6 %. After R. solanacearum inoculation, Si amendments significantly increased the amount of soil bacteria and actinomycetes and reduced soil fungi/soil bacteria ratio by 53.6 %. The results suggested that Si amendment is an effective approach to control R. solanacearum. Moreover, Si-mediated resistance in tomato against R. solanacearum is associated with the changes of soil microorganism amount and soil enzyme activity.

  4. Draft Genome Sequence of Highly Virulent Race 4/Biovar 3 of Ralstonia solanacearum CaRs_Mep Causing Bacterial Wilt in Zingiberaceae Plants in India.

    PubMed

    Kumar, Aundy; Munjal, Vibhuti; Sheoran, Neelam; Prameela, Thekkan Puthiyaveedu; Suseelabhai, Rajamma; Aggarwal, Rashmi; Jain, Rakesh Kumar; Eapen, Santhosh J

    2017-01-05

    The genome of Ralstonia solanacearum CaRs_Mep, a race 4/biovar 3/phylotype I bacterium causing wilt in small cardamom and other Zingiberaceae plants, was sequenced. Analysis of the 5.7-Mb genome sequence will aid in better understanding of the genetic determinants of host range, host jump, survival, pathogenicity, and virulence of race 4 of R. solanacearum.

  5. Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity.

    PubMed

    Lonjon, Fabien; Turner, Marie; Henry, Céline; Rengel, David; Lohou, David; van de Kerkhove, Quitterie; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2016-02-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume

  6. Significant Effects Due to Peptone in Kelman Medium on Colony Characteristics and Virulence of Ralstonia solanacearum in Tomato

    PubMed Central

    Thomas, Pious; Upreti, Reshmi

    2014-01-01

    The study was taken up to assess if the media constituents played any role in governing the variable colony characteristics or pathogenicity of the bacterial wilt pathogen, Ralstonia solanacearum cultured on the widely employed Kelman medium. The effects due to the constituents 2,3,5-triphenyl tetrazolium chloride (TTC), peptone, casein hydrolysate and glucose on colony characteristics were investigated using -80°C stored culture of strain ‘NH-Av01’ (race 1, biovar 3) isolated from tomato. Comparing the pigment inducing TTC from two brands, its source or mode of storage/incorporation did not impart any significant effects. The source of peptone, on the other hand, displayed striking effects on the extent of colony growth, fluidity and red pigmentation depending on type, brand or batch / lot of manufacture as documented with 20 different formulations. Significant differences in the pathogenicity of isolate derived from different peptone sources in seedling-challenge assay on tomato were observed. The observations on peptone effects were endorsed with four other isolates belonging to distinct geographic locations, crops (eggplant, chilli, ginger) or races (race 1 or 4). The peptone source did not influence the pathogen-responses in biovar tests but notably altered the pattern of lawn formation and inhibition zone development during antagonistic assays. Casein hydrolysate displayed some variable effects while glucose source had no effect. This study brings to light the significant modifying effects by the peptone-constituent in Kelman medium on the physiology of R. solanacearum and the virulence of isolate and the need to consider the source of media components during culture maintenance, host-pathogen interaction studies or microbe-microbe interaction investigations. PMID:25408775

  7. rpoN1, but not rpoN2, is required for twitching motility, natural competence, growth on nitrate, and virulence of Ralstonia solanacearum

    PubMed Central

    Ray, Suvendra K.; Kumar, Rahul; Peeters, Nemo; Boucher, Christian; Genin, Stephane

    2015-01-01

    The plant pathogen Ralstonia solanacearum has two genes encoding for the sigma factor σ54: rpoN1, located in the chromosome and rpoN2, located in a distinct “megaplasmid” replicon. In this study, individual mutants as well as a double mutant of rpoN were created in R. solanacearum strain GMI1000 in order to determine the extent of functional overlap between these two genes. By virulence assay we observed that rpoN1 is required for virulence whereas rpoN2 is not. In addition rpoN1 controls other important functions such twitching motility, natural transformation and growth on nitrate, unlike rpoN2. The rpoN1 and rpoN2 genes have different expression pattern, the expression of rpoN1 being constitutive whereas rpoN2 expression is induced in minimal medium and in the presence of plant cells. Moreover, the expression of rpoN2 is dependent upon rpoN1. Our work therefore reveals that the two rpoN genes are not functionally redundant in R. solanacearum. A list of potential σ54 targets was identified in the R. solanacearum genome and suggests that multiple traits are under the control of these regulators. Based on these findings, we provide a model describing the functional connection between RpoN1 and the PehR pathogenicity regulator and their dual role in the control of several R. solanacearum virulence determinants. PMID:25852679

  8. Induction of the viable but nonculturable state of Ralstonia solanacearum by low temperature in the soil microcosm and its resuscitation by catalase.

    PubMed

    Kong, Hyun Gi; Bae, Ju Young; Lee, Hyoung Ju; Joo, Hae Jin; Jung, Eun Joo; Chung, Eunsook; Lee, Seon-Woo

    2014-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state.

  9. Development of variable number of tandem repeats typing schemes for Ralstonia solanacearum, the agent of bacterial wilt, banana Moko disease and potato brown rot.

    PubMed

    N'guessan, Carine Aya; Brisse, Sylvain; Le Roux-Nio, Anne-Claire; Poussier, Stéphane; Koné, Daouda; Wicker, Emmanuel

    2013-03-01

    Ralstonia solanacearum is an important soil borne bacterial plant pathogen causing bacterial wilt on many important crops. To better monitor epidemics, efficient tools that can identify and discriminate populations are needed. In this study, we assessed variable number of tandem repeats (VNTR) genotyping as a new tool for epidemiological surveillance of R. solanacearum phylotypes, and more specifically for the monitoring of the monomorphic ecotypes "Moko" (banana-pathogenic) and "brown rot" (potato-pathogenic under cool conditions). Screening of six R. solanacearum genome sequences lead to select 36 VNTR loci that were preliminarily amplified on 24 strains. From this step, 26 single-locus primer pairs were multiplexed, and applied to a worldwide collection of 337 strains encompassing the whole phylogenetic diversity, with revelation on a capillary-electrophoresis genotype. Four loci were monomorphic within all phylotypes and were not retained; the other loci were highly polymorphic but displayed a clear phylotype-specificity. Phylotype-specific MLVA schemes were thus defined, based on 13 loci for phylotype I, 12 loci for phylotype II, 11 loci for phylotype III and 6 for phylotype IV. MLVA typing was significantly more discriminative than egl-based sequevar typing, particularly on monomorphic "brown rot" ecotype (phylotype IIB/sequevar 1) and "Moko disease" clade 4 (Phylotype IIB/sequevar 4). Our results raise promising prospects for studies of population genetic structures and epidemiological monitoring.

  10. Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions.

    PubMed

    Yim, Woojong; Seshadri, Sundaram; Kim, Kiyoon; Lee, Gillseung; Sa, Tongmin

    2013-06-01

    Bacteria of genus Methylobacterium have been found to promote plant growth and regulate the level of ethylene in crop plants. This work is aimed to test the induction of defense responses in tomato against bacterial wilt by stress ethylene level reduction mediated by the ACC deaminase activity of Methylobacterium strains. Under greenhouse conditions, the disease index value in Methylobacterium sp. inoculated tomato plants was lower than control plants. Plants treated with Methylobacterium sp. challenge inoculated with Ralstonia solanacearum (RS) showed significantly reduced disease symptoms and lowered ethylene emission under greenhouse condition. The ACC and ACO (1-aminocyclopropane-1-carboxylate oxidase) accumulation in tomato leaves were significantly reduced with Methylobacterium strains inoculation. While ACC oxidase gene expression was found higher in plants treated with R. solanacearum than Methylobacterium sp. treatment, PR proteins related to induced systemic resistance like β-1,3-glucanase, PAL, PO and PPO were increased in Methylobacterium sp. inoculated plants. A significant increase in β-1,3-glucanase and PAL gene expression was found in all the Methylobacterium spp. treatments compared to the R. solanacearum treatment. This study confirms the activity of Methylobacterium sp. in increasing the defense enzymes by modulating the ethylene biosynthesis pathway and suggests the use of methylotrophic bacteria as potential biocontrol agents in tomato cultivation.

  11. Genomic characterization of ϕRS603, a filamentous bacteriophage that is infectious to the phytopathogen Ralstonia solanacearum.

    PubMed

    Van, Truong Thi Bich; Yoshida, Shohei; Miki, Kaito; Kondo, Akihiro; Kamei, Kaeko

    2014-12-01

    A filamentous bacteriophage (ϕ), ϕRS603, which is infectious to the phytopathogen Ralstonia solanacearum was isolated. ϕRS603 was found to have a circular single-stranded DNA genome composed of 7679 nucleotides and to contain 13 putative open reading frames (ORFs). The ϕRS603 genome showed strong similarity with those of Ralstonia phages ϕRSM1 and ϕRSM3, as reported by Askora et al. The ϕRS603 genome had no ORFs corresponding to ORFs 2, 3, 13 and 14 (integrase) of ϕRSM3. ϕRS603 had an ORF that was homologous to other Ralstonia phages ϕRSS0 and ϕRSS1; however, ϕRSM1 and ϕRSM3 did not.

  12. Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum.

    PubMed

    Nahar, Kamrun; Matsumoto, Iyo; Taguchi, Fumiko; Inagaki, Yoshishige; Yamamoto, Mikihiro; Toyoda, Kazuhiro; Shiraishi, Tomonori; Ichinose, Yuki; Mukaihara, Takafumi

    2014-04-01

    Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid-resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp-dependent manner. An Agrobacterium-mediated transient expression system revealed that Rip36, a putative Zn-dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn-binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002-derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn-dependent protease motif is essential for this activity.

  13. Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall.

    PubMed

    Digonnet, Catherine; Martinez, Yves; Denancé, Nicolas; Chasseray, Marine; Dabos, Patrick; Ranocha, Philippe; Marco, Yves; Jauneau, Alain; Goffner, Deborah

    2012-11-01

    The compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome.

  14. Contribution of Folate Biosynthesis to Ralstonia solanacearum Proliferation in Intercellular Spaces

    PubMed Central

    Shinohara, Rena; Kanda, Ayami; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2005-01-01

    The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence. PMID:15640216

  15. Draft Genome Sequence of Highly Virulent Race 4/Biovar 3 of Ralstonia solanacearum CaRs_Mep Causing Bacterial Wilt in Zingiberaceae Plants in India

    PubMed Central

    Munjal, Vibhuti; Sheoran, Neelam; Prameela, Thekkan Puthiyaveedu; Suseelabhai, Rajamma; Aggarwal, Rashmi; Jain, Rakesh Kumar; Eapen, Santhosh J.

    2017-01-01

    ABSTRACT The genome of Ralstonia solanacearum CaRs_Mep, a race 4/biovar 3/phylotype I bacterium causing wilt in small cardamom and other Zingiberaceae plants, was sequenced. Analysis of the 5.7-Mb genome sequence will aid in better understanding of the genetic determinants of host range, host jump, survival, pathogenicity, and virulence of race 4 of R. solanacearum. PMID:28057749

  16. Expressed sequence tags in cultivated peanut (Arachis hypogaea): discovery of genes in seed development and response to Ralstonia solanacearum challenge.

    PubMed

    Huang, Jiaquan; Yan, Liying; Lei, Yong; Jiang, Huifang; Ren, Xiaoping; Liao, Boshou

    2012-11-01

    Although an important oil crop, peanut has only 162,030 expressed sequence tags (ESTs) publicly available, 86,943 of which are from cultivated plants. More ESTs from cultivated peanuts are needed for isolation of stress-resistant, tissue-specific and developmentally important genes. Here, we generated 63,234 ESTs from our 5 constructed peanut cDNA libraries of Ralstonia solanacearum challenged roots, R. solanacearum challenged leaves, and unchallenged cultured peanut roots, leaves and developing seeds. Among these ESTs, there were 14,547 unique sequences with 7,961 tentative consensus sequences and 6,586 singletons. Putative functions for 47.8 % of the sequences were identified, including transcription factors, tissue-specific genes, genes involved in fatty acid biosynthesis and oil formation regulation, and resistance gene analogue genes. Additionally, differentially expressed genes, including those involved in ethylene and jasmonic acid signal transduction pathways, from both peanut leaves and roots, were identified in R. solanacearum challenged samples. This large expression dataset from different peanut tissues will be a valuable source for marker development and gene expression analysis. It will also be helpful for finding candidate genes for fatty acid synthesis and oil formation regulation as well as for studying mechanisms of interactions between the peanut host and R. solanacearum pathogen.

  17. PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

    PubMed

    Zhang, Bo; Tremousaygue, Dominique; Denancé, Nicolas; van Esse, H Peter; Hörger, Anja C; Dabos, Patrick; Goffner, Deborah; Thomma, Bart P H J; van der Hoorn, Renier A L; Tuominen, Hannele

    2014-09-01

    PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R.  solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

  18. In Vivo Detection of the Cyclic Osmoregulated Periplasmic Glucan of Ralstonia solanacearum by High-Resolution Magic Angle Spinning NMR

    NASA Astrophysics Data System (ADS)

    Wieruszeski, J.-M.; Bohin, A.; Bohin, J.-P.; Lippens, G.

    2001-07-01

    We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.

  19. Molecular chaperons and co-chaperons, Hsp90, RAR1, and SGT1 negatively regulate bacterial wilt disease caused by Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Ito, Makoto; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2015-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt disease. To better understand the molecular mechanisms involved in interaction between Nicotiana benthamiana and R. solanacearum, we focused on Hsp90, RAR1 and SGT1. Appearances of wilt symptom were significantly suppressed in Hsp90, RAR1 and SGT1-silenced plants compared with control plants. In RAR1-silenced plants, population of R. solanacearum increased in a similar manner to control plants. In contrast, multiplication of R. solanacearum was significantly suppressed in Hsp90 and SGT1-silenced plants. In addition, expression of PR genes were increased in Hsp90 and SGT1-silenced plants challenged with R. solanacearum. Therefore, RAR1 might be required for disease development or suppression of disease tolerance. These results also suggested that Hsp90 and/or SGT1 might play an important role in suppression of plant defenses leading to disease susceptibility and disease development.

  20. Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity*

    PubMed Central

    Lonjon, Fabien; Turner, Marie; Henry, Céline; Rengel, David; Lohou, David; van de Kerkhove, Quitterie; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2016-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume

  1. Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester.

    PubMed

    Flavier, A B; Ganova-Raeva, L M; Schell, M A; Denny, T P

    1997-11-01

    Bacteria employ autoinduction systems to sense the onset of appropriate cell density for expression of developmental genes. In many gram-negative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families. Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium that appears to autoregulate its virulence genes, produces compounds that promote expression of several heterologous acyl-HSL-responsive reporter gene constructs. High-pressure liquid chromatography of highly concentrated ethyl acetate extracts revealed that culture supernatants of strain AW1 contained two compounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL. To investigate the role of these acyl-HSLs in R. solanacearum virulence gene expression, transposon mutants that were deficient for inducing an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were generated. Three loci involved in normal acyl-HSL production were identified, one of which was shown to contain the divergently transcribed solR and solI genes, the luxR and luxI homologs, respectively. A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardless of the locus inactivated) and a naturally acyl-HSL-defective strain of R. solanacearum to produce acyl-HSLs. Inactivation of solI abolished production of all detectable acyl-HSLs but affected neither the expression of virulence genes in culture nor the ability to wilt tomato plants. AW1 has a functional autoinduction system, because (i) expression of solI required SolR and acyl-HSL and (ii) expression of a gene linked to solR and solI, designated aidA, was acyl-HSL dependent. Because AidA has no homologs in the protein databases, its discovery provided no clues as to the role of acyl-HSLs in R. solanacearum gene regulation. However, expression of solR and solI required the global LysR-type virulence regulator PhcA, and both

  2. An evaluation of the wilt-causing bacterium Ralstonia solanacearum as a potential biological control agent for the alien Kahili ginger (Hedychium gardnerianum) in Hawaiian forests

    USGS Publications Warehouse

    1999-01-01

    Kahili ginger (Hedychium gardnerianum) is an invasive weed in tropical forests in Hawaii and elsewhere. Bacterial wilt caused by the ginger strain of Ralstonia(=Pseudomonas) solanacearum systemically infects edible ginger (Zingiber officinale) and ornamental gingers (Hedychium spp.), causing wilt in infected plants. The suitability of R. solanacearum as a biological control agent for kahili ginger was investigated by inoculating seedlings and rooted cuttings of native forest plants, ornamental ginger, and solanaceous species to confirm host specificity. Inoculation via stem injection or root wounding with a bacterial–water suspension was followed by observation for 8 weeks. Inoculations on H. gardnerianum were then carried out in ohia-lehua (Metrosideros polymorpha) wet forests of Hawaii Volcanoes National Park to determine the bacterium's efficacy in the field. No native forest or solanaceous species developed wilt or other symptoms during the study. The bacterium caused limited infection near the inoculation site on H. coronarium, Z. zerumbet, Heliconia latispatha, and Musa sapientum. However, infection did not become systemic in any of these species, and normal growth resumed following appearance of initial symptoms. All inoculated H. gardnerianum plants developed irreversible chlorosis and severe wilting 3–4 weeks following inoculation. Systemic infection also caused death and decay of rhizomes. Most plants were completely dead 16–20 weeks following inoculation. The destructiveness of the ginger strain of R. solanacearum to edible ginger has raised questions regarding its use for biological control. However, because locations of kahili ginger infestations are often remote, the risk of contaminating edible ginger plantings is unlikely. The ability of this bacterium to cause severe disease in H. gardnerianum in the field, together with its lack of virulence in other ginger species, contributes to its potential as a biological control agent.

  3. Effect of Seed Treatment by Cold Plasma on the Resistance of Tomato to Ralstonia solanacearum (Bacterial Wilt)

    PubMed Central

    Jiang, Jiafeng; Lu, Yufang; Li, Jiangang; Li, Ling; He, Xin; Shao, Hanliang; Dong, Yuanhua

    2014-01-01

    This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H2O2) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H2O2 levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW−1, 140% greater than that of the control. Activities of POD (421.3 U gFW−1), PPO (508.8 U gFW−1) and PAL (707.3 U gFW−1) were also greater in the treated plants than in the controls (103.0 U gFW−1, 166.0 U gFW−1 and 309.4 U gFW−1, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H2O2 concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum. PMID:24840508

  4. Effect of seed treatment by cold plasma on the resistance of tomato to Ralstonia solanacearum (Bacterial Wilt).

    PubMed

    Jiang, Jiafeng; Lu, Yufang; Li, Jiangang; Li, Ling; He, Xin; Shao, Hanliang; Dong, Yuanhua

    2014-01-01

    This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H2O2) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H2O2 levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW-1, 140% greater than that of the control. Activities of POD (421.3 U gFW-1), PPO (508.8 U gFW-1) and PAL (707.3 U gFW-1) were also greater in the treated plants than in the controls (103.0 U gFW-1, 166.0 U gFW-1 and 309.4 U gFW-1, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H2O2 concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum.

  5. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids

    PubMed Central

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes’s response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery. PMID:27551287

  6. [Genetic variability of the bacterium Ralstonia solanacearum (Burkholderiales: Burholderiaceae) in the banana-growing region of Uraba (Colombia)].

    PubMed

    Cardozo, Carolina; Rodríguez, Paola; Cotes, José Miguel; Marín, Mauricio

    2010-03-01

    The banana moko disease, caused by the bacterium Ralstonia solanacearum, is one of the most important phytopathological problems of the banana agribusiness in tropical countries. In Uraba and Magdalena (Colombia), the main exporting regions of banana in Colombia, this disease causes a destruction estimated in 16.5 ha/year. The bacterium presents an extremely high level of genetic variation that affects control measures. This is the first study of its variation in Colombia and was done with AFLP molecular markers on a population of 100 isolates from banana plants, soils and "weeds". The high level of genetic diversity, with Nei and Shannon indexes of h=0.32 and I=0.48, respectively, and the AMOVA, showed that this population is subestructured (Fst=0.66): the host is the main factor of differentiation. Even so, previous tests show that all varieties have pathogenicity on Musa.

  7. The C-terminal extension of PrhG impairs its activation of hrp expression and virulence in Ralstonia solanacearum.

    PubMed

    Zhang, Yong; Luo, Feng; Hikichi, Yasufumi; Kiba, Akinori; Yasuo, Igarashi; Ohnishi, Kouhei

    2015-04-01

    Ralstonia solanacearum is the second most destructive bacterial plant pathogens worldwide and HrpG is the master regulator of its pathogenicity. PrhG is a close paralogue of HrpG and both belong to OmpR/PhoB family of two-component response regulators. Despite a high similarity (72% global identity and 96% similarity in helix-loop-helix domain), they display distinct roles in pathogenicity. HrpG is necessary for the bacterial growth in planta and pathogenicity, while PrhG is dispensable for bacterial growth in planta and contributes little to pathogenicity. The main difference between HrpG and PrhG is the 50-amino-acid-long C-terminal extension in PrhG (amino-acid residues 230-283), which is absent in HrpG. When this extension is deleted, truncated PrhGs (under the control of its native promoter) allowed complete recovery of bacterial growth in planta and wild-type virulence of hrpG mutant. This novel finding demonstrates that the extension region in PrhG is responsible for the functional difference between HrpG and PrhG, which may block the binding of PrhG to target promoters and result in impaired activation of hrp expression by PrhG and reduced virulence of R. solanacearum.

  8. The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses.

    PubMed

    Sang, Yuying; Wang, Yaru; Ni, Hong; Cazalé, Anne-Claire; She, Yi-Min; Peeters, Nemo; Macho, Alberto P

    2016-10-21

    The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen-induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ-glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.

  9. The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

    PubMed

    Popa, Crina; Li, Liang; Gil, Sergio; Tatjer, Laura; Hashii, Keisuke; Tabuchi, Mitsuaki; Coll, Núria S; Ariño, Joaquín; Valls, Marc

    2016-06-03

    Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

  10. The involvement of the PilQ secretin of type IV pili in phage infection in Ralstonia solanacearum.

    PubMed

    Narulita, Erlia; Addy, Hardian Susilo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2016-01-22

    PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum.

  11. The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway

    PubMed Central

    Popa, Crina; Li, Liang; Gil, Sergio; Tatjer, Laura; Hashii, Keisuke; Tabuchi, Mitsuaki; Coll, Núria S.; Ariño, Joaquín; Valls, Marc

    2016-01-01

    Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation. PMID:27257085

  12. Phylogenomic analysis of the genus Ralstonia based on 686 single-copy genes.

    PubMed

    Zhang, Yucheng; Qiu, Sai

    2016-01-01

    The genus Ralstonia contains species that are devastating plant pathogens, opportunistic human pathogens, and/or important degraders of xenobiotic and recalcitrant compounds. However, significant nomenclature problems exist, especially for the Ralstonia solanacearum species complex which consists of four phylotypes. Phylogenomics of the Ralstonia genus was investigated via a comprehensive analysis of 39 Ralstonia genomes as well as four genomes of Cupriavidus necator (more commonly known by its previous name Ralstonia eutropha). These data revealed 686 single-copy orthologs that could be extracted from the Ralstonia core-genome and used to reconstruct the phylogeny of the genus Ralstonia. The generated tree has strong bootstrap support for almost all branches. We also estimated the in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) values between each genome. Our data confirmed that whole genome sequence data provides a powerful tool to resolve the complex taxonomic questions of the genus Ralstonia, e.g. strains of Ralstonia solanacearum phylotype IIA and IIB may represent two subspecies of R. solanacearum, and strains of R. solanacearum phylotype I and III may be classified into two subspecies of Ralstonia pseudosolanacearum. Recently, strains of R. solanacearum phylotype IV were proposed to be reclassified into different subspecies of Ralstonia syzygii; our study, however, showed that phylotype IV strains had high isDDH values (83.8-96.1 %), indicating it may be not appropriate to classify these closely related strains into different subspecies. We also evaluated the performance of six chromosomal housekeeping genes (gdhA, mutS, adk, leuS, rplB and gyrB) used in Ralstonia phylogenetic inference. The multilocus sequence analysis of these six marker genes was able to reliably infer the phylogenetic relationships of the genus Ralstonia.

  13. An RpoS (sigmaS) homologue regulates acylhomoserine lactone-dependent autoinduction in Ralstonia solanacearum.

    PubMed

    Flavier, A B; Schell, M A; Denny, T P

    1998-05-01

    Many bacteria sense an appropriate growth condition or a critical population density for gene expression by producing acylhomoserine lactones (acyl-HSLs) that act as intercellular autoinduction signals. We recently showed that, in Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium, acyl-HSL production requires soll, which encodes a putative acyl-HSL synthase, and that its expression is positively regulated by the acyl-HSL-responsive SolR transcriptional regulator. This acyl-HSL-dependent autoinduction system is noteworthy because (i) it is regulated by a 'higher level' autoinducer system (responsive to 3-hydroxypalmitic acid methyl ester) via PhcA, a LysR-type transcriptional regulator and (ii) acyl-HSL production requires two additional unlinked loci. As reported here, cloning and sequencing of one of these other loci revealed that it encodes a homologue of RpoS, an alternative sigma factor (sigmaS) that in other bacteria activates gene expression during stationary phase or in response to stress conditions. R. solanacearum RpoS (RpoS(Rso)) was demonstrated to function as a sigma factor because when introduced in trans into an Escherichia coli rpoS mutant it largely restored expression of the RpoS-dependent bolAp1 gene. Mutation of rpoS(Rso) in R. solanacearum reduced survival during starvation and low pH conditions, but did not affect survival during exposure to hydrogen peroxide, high osmolarity or high temperature. This mutant was also altered in its production of several virulence factors and wilted tomato plants several days more slowly than the wild-type parent. Transcription of solR and soll were decreased in an rpoS(Rso) background (thereby reducing acyl-HSL production), but neither mutations in solR, soll or phcA nor addition of acyl-HSLs affected rpoS(Rso) expression. Therefore, in R. solanacearum the acyl-HSL-dependent autoinduction system is controlled both by a second autoinduction system and by the RpoS(Rso) sigma factor.

  14. Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment.

    PubMed

    Tans-Kersten, Julie; Brown, Darby; Allen, Caitilyn

    2004-06-01

    Swimming motility allows the bacterial wilt pathogen Ralstonia solanacearum to efficiently invade and colonize host plants. However, the bacteria are essentially nonmotile once inside plant xylem vessels. To determine how and when motility genes are expressed, we cloned and mutated flhDC, which encodes a major regulator of flagellar biosynthesis and bacterial motility. An flhDC mutant was nonmotile and less virulent than its wild-type parent on both tomato and Arabidopsis; on Arabidopsis, the flhDC mutant also was less virulent than a nonmotile fliC flagellin mutant. Genes in the R. solanacearum motility regulon had strikingly different expression patterns in culture and in the plant. In culture, as expected, flhDC expression depended on PehSR, a regulator of early virulence factors; and, in turn, FlhDC was required for fliC (flagellin) expression. However, when bacteria grew in tomato plants, flhDC was expressed in both wild-type and pehR mutant backgrounds, although PehSR is necessary for motility both in culture and in planta. Both flhDC and pehSR were significantly induced in planta relative to expression levels in culture. Unexpectedly, the fliC gene was expressed in planta at cell densities where motile bacteria were not observed, as well as in a nonmotile flhDC mutant. Thus, expression of flhDC and flagellin itself are uncoupled from bacterial motility in the host environment, indicating that additional signals and regulatory circuits repress motility during plant pathogenesis.

  15. Overexpression of a Chinese cabbage BrERF11 transcription factor enhances disease resistance to Ralstonia solanacearum in tobacco.

    PubMed

    Lai, Yan; Dang, Fengfeng; Lin, Jing; Yu, Lu; Shi, Youliang; Xiao, Yuhua; Huang, Mukun; Lin, Jinhui; Chen, Chengcong; Qi, Aihua; Liu, Zhiqin; Guan, Deyi; Mou, Shaoliang; Qiu, Ailian; He, Shuilin

    2013-01-01

    Ethylene-responsive factors (ERFs) play diverse roles in plant growth, developmental processes and stress responses. However, the roles and underlying mechanism of ERFs remain poorly understood, especially in non-model plants. In this study, a full length cDNA of ERF gene was isolated from the cDNA library of Chinese cabbage. According to sequence alignment, we found a highly conservative AP2/ERF domain, two nuclear localization signals, and an ERF-associated Amphiphilic Repression (EAR) motif in its C-terminal region. It belonged to VIIIa group ERFs sharing the highest sequence identity with AtERF11 in all of the ERFs in Arabidopsis and designated BrERF11. BrERF11-green fluorescence protein (GFP) transient expressed in onion epidermis cells localized to the nucleus. The transcript levels of BrERF11 were induced by exogenous salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), and hydrogen peroxide (H(2)O(2)). Constitutive expression of BrERF11 enhanced tolerance to Ralstonia solanacearum infection in transgenic tobacco plants, which was coupled with hypersensitive response (HR), burst of H(2)O(2) and upregulation of defense-related genes including HR marker genes, SA-, JA-dependent pathogen-related genes and ET biosynthesis associated genes and downregulation of CAT1, suggesting BrERF11 may participate in pathogen-associated molecular pattern (PAMP)- and effector-triggered immunity (PTI and ETI) mediated by SA-, JA- and ET-dependent signaling mechanisms.

  16. Bacillus thuringiensis suppresses bacterial wilt disease caused by Ralstonia solanacearum with systemic induction of defense-related gene expression in tomato.

    PubMed

    Hyakumachi, Mitsuro; Nishimura, Mitsuyoshi; Arakawa, Tatsuyuki; Asano, Shinichiro; Yoshida, Shigenobu; Tsushima, Seiya; Takahashi, Hideki

    2013-01-01

    Bacillus thuringiensis is a naturally abundant Gram-positive bacterium and a well-known, effective bio-insecticide. Recently, B. thuringiensis has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. In this study, the bacterial wilt disease-suppressing activity of B. thuringiensis was examined in tomato plants. Treatment of tomato roots with B. thuringiensis culture followed by challenge inoculation with Ralstonia solanacearum suppressed the development of wilt symptoms to less than one third of the control. This disease suppression in tomato plants was reproduced by pretreating their roots with a cell-free filtrate (CF) that had been fractionated from B. thuringiensis culture by centrifugation and filtration. In tomato plants challenge-inoculated with R. solanacearum after pretreatment with CF, the growth of R. solanacearum in stem tissues clearly decreased, and expression of defense-related genes such as PR-1, acidic chitinase, and β-1,3-glucanase was induced in stem and leaf tissues. Furthermore, the stem tissues of tomato plants with their roots were pretreated with CF exhibited resistance against direct inoculation with R. solanacearum. Taken together, these results suggest that treatment of tomato roots with the CF of B. thuringiensis systemically suppresses bacterial wilt through systemic activation of the plant defense system.

  17. Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

    PubMed

    Denancé, Nicolas; Ranocha, Philippe; Oria, Nicolas; Barlet, Xavier; Rivière, Marie-Pierre; Yadeta, Koste A; Hoffmann, Laurent; Perreau, François; Clément, Gilles; Maia-Grondard, Alessandra; van den Berg, Grardy C M; Savelli, Bruno; Fournier, Sylvie; Aubert, Yann; Pelletier, Sandra; Thomma, Bart P H J; Molina, Antonio; Jouanin, Lise; Marco, Yves; Goffner, Deborah

    2013-01-01

    Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

  18. Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum.

    PubMed

    Herlache, T C; Hotchkiss, A T; Burr, T J; Collmer, A

    1997-01-01

    DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum. Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA. Mature PehA accumulated primarily in the periplasm of A. vitis and pehA+ Escherichia coli cells during exponential growth. A. vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E. carotovora and R. solanacearum polygalacturonases.

  19. Constraints on Genome Dynamics Revealed from Gene Distribution among the Ralstonia solanacearum Species

    PubMed Central

    Lefeuvre, Pierre; Cellier, Gilles; Remenant, Benoît; Chiroleu, Frédéric; Prior, Philippe

    2013-01-01

    Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens. PMID:23723974

  20. A Resource Allocation Trade-Off between Virulence and Proliferation Drives Metabolic Versatility in the Plant Pathogen Ralstonia solanacearum

    PubMed Central

    Marmiesse, Lucas; Gouzy, Jérôme

    2016-01-01

    Bacterial pathogenicity relies on a proficient metabolism and there is increasing evidence that metabolic adaptation to exploit host resources is a key property of infectious organisms. In many cases, colonization by the pathogen also implies an intensive multiplication and the necessity to produce a large array of virulence factors, which may represent a significant cost for the pathogen. We describe here the existence of a resource allocation trade-off mechanism in the plant pathogen R. solanacearum. We generated a genome-scale reconstruction of the metabolic network of R. solanacearum, together with a macromolecule network module accounting for the production and secretion of hundreds of virulence determinants. By using a combination of constraint-based modeling and metabolic flux analyses, we quantified the metabolic cost for production of exopolysaccharides, which are critical for disease symptom production, and other virulence factors. We demonstrated that this trade-off between virulence factor production and bacterial proliferation is controlled by the quorum-sensing-dependent regulatory protein PhcA. A phcA mutant is avirulent but has a better growth rate than the wild-type strain. Moreover, a phcA mutant has an expanded metabolic versatility, being able to metabolize 17 substrates more than the wild-type. Model predictions indicate that metabolic pathways are optimally oriented towards proliferation in a phcA mutant and we show that this enhanced metabolic versatility in phcA mutants is to a large extent a consequence of not paying the cost for virulence. This analysis allowed identifying candidate metabolic substrates having a substantial impact on bacterial growth during infection. Interestingly, the substrates supporting well both production of virulence factors and growth are those found in higher amount within the plant host. These findings also provide an explanatory basis to the well-known emergence of avirulent variants in R. solanacearum

  1. Differential occurrence of oxidative burst and antioxidative mechanism in compatible and incompatible interactions of Solanum lycopersicum and Ralstonia solanacearum.

    PubMed

    Mandal, Sudhamoy; Das, Rupa Kumar; Mishra, Sanjeev

    2011-02-01

    Striking increase in reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) has been demonstrated to occur in plants in response to pathogen attack. The aim of this study was to investigate the biochemical aspects of ROS generation, antioxidative mechanism and cell wall reinforcement as responses of tomato cultivars Arka Meghali (AM; susceptible) and BT-10 (BT; resistant) against Ralstonia solanacearum (Ralsol). While the oxidative burst was characterized by a single phase ROS increase in AM, there was a clear bi-phasic ROS generation in BT. The first significant increase of H(2)O(2) production was noticed at 12 h post-inoculation (hpi) followed by a sharp increase in H(2)O(2) generation after 36 hpi. Lipid peroxidation was more in roots of AM than that of BT after pathogen inoculation. Superoxide dismutase and catalase activities were continuously at very high level in Ralsol-inoculated BT plants, whereas activities of the enzymes were observed to decrease at later stage in Ralsol-inoculated AM plants. Guaiacol peroxidase activity was high in Ralsol-inoculated roots of both cultivars, but BT recorded much higher activity than AM. Higher activity of ascorbate peroxidase in inoculated BT might be an indication of better scavenging activity of the enzyme. Total phenolic content and lignin deposition were significantly higher in Ralsol-inoculated BT compared to inoculated AM. Our results indicate that increased level of ROS production coupled with more efficient antioxidative system, lower rate of lipid peroxidation and high lignin deposition in cell wall may contribute to the resistance of tomato plants to Ralsol.

  2. Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects

    PubMed Central

    Upreti, Reshmi; Thomas, Pious

    2015-01-01

    This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8–9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen

  3. CaWRKY6 transcriptionally activates CaWRKY40, regulates Ralstonia solanacearum resistance, and confers high-temperature and high-humidity tolerance in pepper.

    PubMed

    Cai, Hanyang; Yang, Sheng; Yan, Yan; Xiao, Zhuoli; Cheng, Junbin; Wu, Ji; Qiu, Ailian; Lai, Yan; Mou, Shaoliang; Guan, Deyi; Huang, Ronghua; He, Shuilin

    2015-06-01

    High temperature (HT), high humidity (HH), and pathogen infection often co-occur and negatively affect plant growth. However, these stress factors and plant responses are generally studied in isolation. The mechanisms of synergistic responses to combined stresses are poorly understood. We isolated the subgroup IIb WRKY family member CaWRKY6 from Capsicum annuum and performed quantitative real-time PCR analysis. CaWRKY6 expression was upregulated by individual or simultaneous treatment with HT, HH, combined HT and HH (HTHH), and Ralstonia solanacearum inoculation, and responded to exogenous application of jasmonic acid (JA), ethephon, and abscisic acid (ABA). Virus-induced gene silencing of CaWRKY6 enhanced pepper plant susceptibility to R. solanacearum and HTHH, and downregulated the hypersensitive response (HR), JA-, ethylene (ET)-, and ABA-induced marker gene expression, and thermotolerance-associated expression of CaHSP24, ER-small CaSHP, and Chl-small CaHSP. CaWRKY6 overexpression in pepper attenuated the HTHH-induced suppression of resistance to R. solanacearum infection. CaWRKY6 bound to and activated the CaWRKY40 promoter in planta, which is a pepper WRKY that regulates heat-stress tolerance and R. solanacearum resistance. CaWRKY40 silencing significantly blocked HR-induced cell death and reduced transcriptional expression of CaWRKY40. These data suggest that CaWRKY6 is a positive regulator of R. solanacearum resistance and heat-stress tolerance, which occurs in part by activating CaWRKY40.

  4. Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco.

    PubMed

    Zhang, Chong; Chen, Hua; Cai, Tiecheng; Deng, Ye; Zhuang, Ruirong; Zhang, Ning; Zeng, Yuanhuan; Zheng, Yixiong; Tang, Ronghua; Pan, Ronglong; Zhuang, Weijian

    2017-01-01

    Bacterial wilt caused by Ralstonia solanacearum is a ruinous soilborne disease affecting more than 450 plant species. Efficient control methods for this disease remain unavailable to date. This study characterized a novel nucleotide-binding site-leucine-rich repeat resistance gene AhRRS5 from peanut, which was up-regulated in both resistant and susceptible peanut cultivars in response to R. solanacearum. The product of AhRRS5 was localized in the nucleus. Furthermore, treatment with phytohormones such as salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (ET) increased the transcript level of AhRRS5 with diverse responses between resistant and susceptible peanuts. Abiotic stresses such as drought and cold conditions also changed AhRRS5 expression. Moreover, transient overexpression induced hypersensitive response in Nicotiana benthamiana. Overexpression of AhRRS5 significantly enhanced the resistance of heterogeneous tobacco to R. solanacearum, with diverse resistance levels in different transgenic lines. Several defence-responsive marker genes in hypersensitive response, including SA, JA and ET signals, were considerably up-regulated in the transgenic lines as compared with the wild type inoculated with R. solanacearum. Nonexpressor of pathogenesis-related gene 1 (NPR1) and non-race-specific disease resistance 1 were also up-regulated in response to the pathogen. These results indicate that AhRRS5 participates in the defence response to R. solanacearum through the crosstalk of multiple signalling pathways and the involvement of NPR1 and R gene signals for its resistance. This study may guide the resistance enhancement of peanut and other economic crops to bacterial wilt disease.

  5. A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences.

    PubMed

    Kumar, Aundy; Prameela, Thekkan Puthiyaveedu; Suseelabhai, Rajamma

    2013-06-01

    Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.

  6. Meta-analysis Reveals That the Genus Pseudomonas Can Be a Better Choice of Biological Control Agent against Bacterial Wilt Disease Caused by Ralstonia solanacearum

    PubMed Central

    Chandrasekaran, Murugesan; Subramanian, Dharaneedharan; Yoon, Ee; Kwon, Taehoon; Chun, Se-Chul

    2016-01-01

    Biological control agents (BCAs) from different microbial taxa are increasingly used to control bacterial wilt caused by Ralstonia solanacearum. However, a quantitative research synthesis has not been conducted on the role of BCAs in disease suppression. Therefore, the present study aimed to meta-analyze the impacts of BCAs on both Ralstonia wilt disease suppression and plant (host) growth promotion. The analysis showed that the extent of disease suppression by BCAs varied widely among studies, with effect size (log response ratio) ranging from −2.84 to 2.13. The disease incidence and severity were significantly decreased on average by 53.7% and 49.3%, respectively. BCAs inoculation also significantly increased fresh and dry weight by 34.4% and 36.1%, respectively on average. Also, BCAs inoculation significantly increased plant yield by 66%. Mean effect sizes for genus Pseudomonas sp. as BCAs were higher than for genus Bacillus spp. Among antagonists tested, P. fluorescens, P. putida, B. cereus, B. subtilis and B. amyloliquefaciens were found to be more effective in general for disease reduction. Across studies, highest disease control was found for P. fluorescens, annual plants, co-inoculation with more than one BCA, soil drench and greenhouse condition were found to be essential in understanding plant responses to R. solanacearum. Our results suggest that more efforts should be devoted to harnessing the potential beneficial effects of these antagonists, not just for plant growth promoting traits but also in mode of applications, BCAs formulations and their field studies should be considered in the future for R. solanacearum wilt disease suppression. PMID:27298597

  7. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    PubMed

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  8. Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection.

    PubMed

    Dang, Fengfeng; Wang, Yuna; She, Jianju; Lei, Yufen; Liu, Zhiqin; Eulgem, Thomas; Lai, Yan; Lin, Jing; Yu, Lu; Lei, Dan; Guan, Deyi; Li, Xia; Yuan, Qian; He, Shuilin

    2014-03-01

    WRKY proteins are encoded by a large gene family and are linked to many biological processes across a range of plant species. The functions and underlying mechanisms of WRKY proteins have been investigated primarily in model plants such as Arabidopsis and rice. The roles of these transcription factors in non-model plants, including pepper and other Solanaceae, are poorly understood. Here, we characterize the expression and function of a subgroup IIe WRKY protein from pepper (Capsicum annuum), denoted as CaWRKY27. The protein localized to nuclei and activated the transcription of a reporter GUS gene construct driven by the 35S promoter that contained two copies of the W-box in its proximal upstream region. Inoculation of pepper cultivars with Ralstonia solanacearum induced the expression of CaWRKY27 transcript in 76a, a bacterial wilt-resistant pepper cultivar, whereas it downregulated the expression of CaWRKY27 transcript in Gui-1-3, a bacterial wilt-susceptible pepper cultivar. CaWRKY27 transcript levels were also increased by treatments with salicylic acid (SA), methyl jasmonate (MeJA) and ethephon (ETH). Transgenic tobacco plants overexpressing CaWRKY27 exhibited resistance to R. solanacearum infection compared to that of wild-type plants. This resistance was coupled with increased transcript levels in a number of marker genes, including hypersensitive response genes, and SA-, JA- and ET-associated genes. By contrast, virus-induced gene silencing (VIGS) of CaWRKY27 increased the susceptibility of pepper plants to R. solanacearum infection. These results suggest that CaWRKY27 acts as a positive regulator in tobacco resistance responses to R. solanacearum infection through modulation of SA-, JA- and ET-mediated signaling pathways.

  9. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000

    PubMed Central

    Lundgren, Benjamin R.; Connolly, Morgan P.; Choudhary, Pratibha; Brookins-Little, Tiffany S.; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T.

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000. PMID:26659655

  10. Effect of calcium cyanamide, ammonium bicarbonate and lime mixture, and ammonia water on survival of Ralstonia solanacearum and microbial community

    NASA Astrophysics Data System (ADS)

    Liu, Lijuan; Sun, Chengliang; Liu, Xingxing; He, Xiaolin; Liu, Miao; Wu, Hao; Tang, Caixian; Jin, Chongwei; Zhang, Yongsong

    2016-01-01

    The inorganic nitrogenous amendments calcium cyanamide (CC), ammonia water (AW), and a mixture of ammonium bicarbonate with lime (A+L) are popularly used as fumigants to control soil-borne disease in China. However, it is unclear which of these fumigants is more effective in controlling R. solanacearum. This present study compared the efficiencies of the three nitrogenous amendments listed above at four nitrogen levels in suppressing the survival of R. solanacearum in soil. The CC showed the best ability to suppress R. solanacearum due to its highest capacity to increase soil and NO2‑ contents and pH. However, AW was more suitable to controlling bacterial wilt caused by R. solanacearum because it had a lower cost and its application rate of 0.25 g N kg‑1 soil could effectively suppress the survival of R. solanacearum. Additionally, soil microbial activity and community populations were restored to their initial state four weeks after the application of each fumigant, indicating that the three fumigants had few detrimental impacts on soil microbial activity and community structure with an exception of the suppression of R. solanacearum. The present study provides guidance for the selection of a suitable alkaline nitrogenous amendment and its application rate in controlling bacterial wilt.

  11. Effect of calcium cyanamide, ammonium bicarbonate and lime mixture, and ammonia water on survival of Ralstonia solanacearum and microbial community

    PubMed Central

    Liu, Lijuan; Sun, Chengliang; Liu, Xingxing; He, Xiaolin; Liu, Miao; Wu, Hao; Tang, Caixian; Jin, Chongwei; Zhang, Yongsong

    2016-01-01

    The inorganic nitrogenous amendments calcium cyanamide (CC), ammonia water (AW), and a mixture of ammonium bicarbonate with lime (A+L) are popularly used as fumigants to control soil-borne disease in China. However, it is unclear which of these fumigants is more effective in controlling R. solanacearum. This present study compared the efficiencies of the three nitrogenous amendments listed above at four nitrogen levels in suppressing the survival of R. solanacearum in soil. The CC showed the best ability to suppress R. solanacearum due to its highest capacity to increase soil and NO2− contents and pH. However, AW was more suitable to controlling bacterial wilt caused by R. solanacearum because it had a lower cost and its application rate of 0.25 g N kg−1 soil could effectively suppress the survival of R. solanacearum. Additionally, soil microbial activity and community populations were restored to their initial state four weeks after the application of each fumigant, indicating that the three fumigants had few detrimental impacts on soil microbial activity and community structure with an exception of the suppression of R. solanacearum. The present study provides guidance for the selection of a suitable alkaline nitrogenous amendment and its application rate in controlling bacterial wilt. PMID:26738601

  12. Effect of calcium cyanamide, ammonium bicarbonate and lime mixture, and ammonia water on survival of Ralstonia solanacearum and microbial community.

    PubMed

    Liu, Lijuan; Sun, Chengliang; Liu, Xingxing; He, Xiaolin; Liu, Miao; Wu, Hao; Tang, Caixian; Jin, Chongwei; Zhang, Yongsong

    2016-01-07

    The inorganic nitrogenous amendments calcium cyanamide (CC), ammonia water (AW), and a mixture of ammonium bicarbonate with lime (A+L) are popularly used as fumigants to control soil-borne disease in China. However, it is unclear which of these fumigants is more effective in controlling R. solanacearum. This present study compared the efficiencies of the three nitrogenous amendments listed above at four nitrogen levels in suppressing the survival of R. solanacearum in soil. The CC showed the best ability to suppress R. solanacearum due to its highest capacity to increase soil and NO2(-) contents and pH. However, AW was more suitable to controlling bacterial wilt caused by R. solanacearum because it had a lower cost and its application rate of 0.25 g N kg(-1) soil could effectively suppress the survival of R. solanacearum. Additionally, soil microbial activity and community populations were restored to their initial state four weeks after the application of each fumigant, indicating that the three fumigants had few detrimental impacts on soil microbial activity and community structure with an exception of the suppression of R. solanacearum. The present study provides guidance for the selection of a suitable alkaline nitrogenous amendment and its application rate in controlling bacterial wilt.

  13. The symbiotic transcription factor MtEFD and cytokinins are positively acting in the Medicago truncatula and Ralstonia solanacearum pathogenic interaction.

    PubMed

    Moreau, Sandra; Fromentin, Justine; Vailleau, Fabienne; Vernié, Tatiana; Huguet, Stéphanie; Balzergue, Sandrine; Frugier, Florian; Gamas, Pascal; Jardinaud, Marie-Françoise

    2014-03-01

    • A plant-microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont Sinorhizobium meliloti. • Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts. • Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor. • The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.

  14. HpaP modulates type III effector secretion in Ralstonia solanacearum and harbours a substrate specificity switch domain essential for virulence.

    PubMed

    Lohou, David; Turner, Marie; Lonjon, Fabien; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2014-08-01

    Many pathogenic bacteria have evolved a type III secretion system (T3SS) to successfully invade their host. This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. T3Es are virulence factors that have been shown to interfere with the host's immunity or to provide nutrients from the host to the bacteria. The Gram-negative bacterium Ralstonia solanacearum is a worldwide major crop pest whose virulence strongly relies on the T3SS. In R. solanacearum, transcriptional regulation has been extensively studied. However, very few data are available concerning the role played by type III-associated regulators, such as type III chaperones and T3SS control proteins. Here, we characterized HpaP, a putative type III secretion substrate specificity switch (T3S4) protein of R. solanacearum which is not secreted by the bacterium or translocated in the plant cells. HpaP self-interacts and interacts with the PopP1 T3E. HpaP modulates the secretion of early (HrpY pilin) and late (AvrA and PopP1 T3Es) type III substrates. HpaP is dispensable for the translocation of T3Es into the host cells. Finally, we identified two regions of five amino acids in the T3S4 domain that are essential for efficient PopP1 secretion and for HpaP's role in virulence on tomato and Arabidopsis thaliana, but not required for HpaP-HpaP and HpaP-PopP1 interactions. Taken together, our results indicate that HpaP is a putative R. solanacearum T3S4 protein important for full pathogenicity on several hosts, acting as a helper for PopP1 secretion, and repressing AvrA and HrpY secretion.

  15. Sensitive quantitative detection of Ralstonia solanacearum in soil by the most probable number-polymerase chain reaction (MPN-PCR) method.

    PubMed

    Inoue, Yasuhiro; Nakaho, Kazuhiro

    2014-05-01

    We developed a sensitive quantitative assay for detecting Ralstonia solanacearum in soil by most probable number (MPN) analysis based on bio-PCR results. For development of the detection method, we optimized an elution buffer containing 5 g/L skim milk for extracting bacteria from soil and reducing contamination of polymerase inhibitors in soil extracts. Because R. solanacearum can grow in water without any added nutrients, we used a cultivation buffer in the culture step of the bio-PCR that contained only the buffer and antibiotics to suppress the growth of other soil microorganisms. To quantify the bacterial population in soil, the elution buffer was added to 10 g soil on a dry weight basis so that the combined weight of buffer, soil, and soil-water was 50 g; 5 mL of soil extract was assumed to originate from 1 g of soil. The soil extract was divided into triplicate aliquots each of 5 mL and 500, 50, and 5 μL. Each aliquot was diluted with the cultivation buffer and incubated at 35 °C for about 24 h. After incubation, 5 μL of culture was directly used for nested PCR. The number of aliquots showing positive results was collectively checked against the MPN table. The method could quantify bacterial populations in soil down to 3 cfu/10 g dried soil and was successfully applied to several types of soil. We applied the method for the quantitative detection of R. solanacearum in horticultural soils, which could quantitatively detect small populations (9.3 cfu/g), but the semiselective media were not able to detect the bacteria.

  16. Enhanced in planta Fitness through Adaptive Mutations in EfpR, a Dual Regulator of Virulence and Metabolic Functions in the Plant Pathogen Ralstonia solanacearum

    PubMed Central

    Rengel, David; Barlet, Xavier; Gouzy, Jérôme

    2016-01-01

    Experimental evolution of the plant pathogen Ralstonia solanacearum, where bacteria were maintained on plant lineages for more than 300 generations, revealed that several independent single mutations in the efpR gene from populations propagated on beans were associated with fitness gain on bean. In the present work, novel allelic efpR variants were isolated from populations propagated on other plant species, thus suggesting that mutations in efpR were not solely associated to a fitness gain on bean, but also on additional hosts. A transcriptomic profiling and phenotypic characterization of the efpR deleted mutant showed that EfpR acts as a global catabolic repressor, directly or indirectly down-regulating the expression of multiple metabolic pathways. EfpR also controls virulence traits such as exopolysaccharide production, swimming and twitching motilities and deletion of efpR leads to reduced virulence on tomato plants after soil drenching inoculation. We studied the impact of the single mutations that occurred in efpR during experimental evolution and found that these allelic mutants displayed phenotypic characteristics similar to the deletion mutant, although not behaving as complete loss-of-function mutants. These adaptive mutations therefore strongly affected the function of efpR, leading to an expanded metabolic versatility that should benefit to the evolved clones. Altogether, these results indicated that EfpR is a novel central player of the R. solanacearum virulence regulatory network. Independent mutations therefore appeared during experimental evolution in the evolved clones, on a crucial node of this network, to favor adaptation to host vascular tissues through regulatory and metabolic rewiring. PMID:27911943

  17. Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt

    PubMed Central

    Tahir, Hafiz Abdul Samad; Gu, Qin; Wu, Huijun; Niu, Yuedi; Huo, Rong; Gao, Xuewen

    2017-01-01

    Volatile organic compounds (VOCs) produced by various bacteria have significant potential to enhance plant growth and to control phytopathogens. Six of the most effective antagonistic Bacillus spp. were used in this study against Ralstonia solanacearum (Rsc) TBBS1, the causal agent of bacterial wilt disease in tobacco. Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22 had the strongest inhibitory effect against Rsc. Thirteen VOCs produced by FZB42 and 10 by LSSC22 were identified using gas chromatography-mass spectrometry analysis. Benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene significantly inhibited the colony size, cell viability, and motility of pathogens and negatively influenced chemotaxis. Transmission and scanning electron microscopy revealed severe morphological and ultra-structural changes in cells of Rsc. Furthermore, VOCs altered the transcriptional expression level of PhcA (a global virulence regulator), type III secretion system (T3SS), type IV secretion system (T4SS), extracellular polysaccharides and chemotaxis-related genes, which are major contributors to pathogenicity, resulting in decreased wilt disease. The VOCs significantly up-regulated the expression of genes related to wilt resistance and pathogen defense. Over-expression of EDS1 and NPR1 suggest the involvement of SA pathway in induction of systemic resistance. Our findings provide new insights regarding the potential of antibacterial VOCs as a biocontrol tool against bacterial wilt diseases. PMID:28091587

  18. Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against Its Core Effectors.

    PubMed

    Clarke, Christopher R; Studholme, David J; Hayes, Byron; Runde, Brendan; Weisberg, Alexandra; Cai, Rongman; Wroblewski, Tadeusz; Daunay, Marie-Christine; Wicker, Emmanuel; Castillo, Jose A; Vinatzer, Boris A

    2015-05-01

    Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.

  19. Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt.

    PubMed

    Tahir, Hafiz Abdul Samad; Gu, Qin; Wu, Huijun; Niu, Yuedi; Huo, Rong; Gao, Xuewen

    2017-01-16

    Volatile organic compounds (VOCs) produced by various bacteria have significant potential to enhance plant growth and to control phytopathogens. Six of the most effective antagonistic Bacillus spp. were used in this study against Ralstonia solanacearum (Rsc) TBBS1, the causal agent of bacterial wilt disease in tobacco. Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22 had the strongest inhibitory effect against Rsc. Thirteen VOCs produced by FZB42 and 10 by LSSC22 were identified using gas chromatography-mass spectrometry analysis. Benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene significantly inhibited the colony size, cell viability, and motility of pathogens and negatively influenced chemotaxis. Transmission and scanning electron microscopy revealed severe morphological and ultra-structural changes in cells of Rsc. Furthermore, VOCs altered the transcriptional expression level of PhcA (a global virulence regulator), type III secretion system (T3SS), type IV secretion system (T4SS), extracellular polysaccharides and chemotaxis-related genes, which are major contributors to pathogenicity, resulting in decreased wilt disease. The VOCs significantly up-regulated the expression of genes related to wilt resistance and pathogen defense. Over-expression of EDS1 and NPR1 suggest the involvement of SA pathway in induction of systemic resistance. Our findings provide new insights regarding the potential of antibacterial VOCs as a biocontrol tool against bacterial wilt diseases.

  20. A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection, pathogen associated molecular patterns and effector molecules and involved in plant immunity.

    PubMed

    Kiba, Akinori; Nakano, Masahito; Vincent-Pope, Patrick; Takahashi, Hirotaka; Sawasaki, Tatsuya; Endo, Yaeta; Ohnishi, Kouhei; Yoshioka, Hirofumi; Hikichi, Yasufumi

    2012-07-01

    To elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with Ralstonia solanacearum. Here, we report the characterization of Nicotiana benthamiana belonging to the SEC14-gene superfamily designated as Nicotiana benthamiana SEC14 (NbSEC14). NbSEC14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant NbSec14 protein had phospholipids transfer activity. NbSEC14 expression was up-regulated in N. benthamiana leaves after inoculation with virulent or avirulent R. solanacearum. Expression of NbSEC14 was induced by treatment with chitin, flg22, and by Agrobacterium-mediated transient expression of INF1 elicitin, AvrA from R. solanacearum, and co-expression of the capsid protein from Tobacco mild green mosaic virus with its cognate resistance L1 protein. NbSEC14-silenced plants showed accelerated growth of both the virulent and avirulent R. solanacearum as well as acceleration of disease development. This study may provide useful information for the further analysis of the function of plant Sec14 protein homologs in the regulation of plant immune responses.

  1. CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

    PubMed Central

    Shen, Lei; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Cheng, Wei; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; Liu, Zhiqin; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-01-01

    CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity. PMID:26928570

  2. Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato.

    PubMed

    Pan, I-Chun; Li, Chia-Wen; Su, Ruey-Chih; Cheng, Chiu-Ping; Lin, Choun-Sea; Chan, Ming-Tsair

    2010-10-01

    Ethylene-responsive transcription factors (ERFs) bind specifically to cis-acting DNA regulatory elements such as GCC boxes and play an important role in the regulation of defense- and stress-related genes in plants. In contrast to other ERFs, class II ERFs contain an ERF-associated amphiphilic repression (EAR) domain and act as GCC-mediated transcriptional repressors. In this study, SlERF3, a class II ERF was isolated from tomato and characterized. To examine whether the EAR motif of class II ERF proteins participates in ERF-mediated functions in plants, the EAR domain was deleted to generate SlERF3ΔRD. We show that SlERF3ΔRD protein retains the character of a transcription factor and becomes a GCC-mediated transcriptional activator. Constitutive expression of full-length SlERF3 in tomato severely suppressed growth and, as a result, no transgenic plants were obtained. However, no apparent effects on growth and development of SlERF3ΔRD transgenic plants were observed. Overexpression of SlERF3ΔRD in transgenic tomato induced expression of pathogenesis-related protein genes such as PR1, PR2 and PR5, and enhanced tolerance to Ralstonia solanacearum. Furthermore, transgenic Arabidopsis and tomatoes constitutively expressing SlERF3ΔRD exhibited reduced levels of membrane lipid peroxidation and enhanced tolerance to salt stress. In comparison with wild-type plants grown under stress conditions, transgenic SlERF3ΔRD tomatoes produced more flowers, fruits, and seeds. This study illustrates a gene-enhancing tolerance to both biotic and abiotic stresses in transgenic plants with the deletion of a repressor domain. Our findings suggest that class II ERF proteins may find important use in crop improvement or genetic engineering to increase stress tolerance in plants.

  3. Draft Genome Sequence for Ralstonia sp. Strain OR214, a Bacterium with Potential for Bioremediation

    PubMed Central

    Utturkar, Sagar M.; Brzoska, Ryann M.; Klingeman, Dawn M.; Epstein, Slava E.; Palumbo, Anthony V.

    2013-01-01

    Ralstonia sp. strain OR214 belongs to the class Betaproteobacteria and was isolated from subsurface sediments in Oak Ridge, TN. A member of this genus has been described as a potential bioremediation agent. Strain OR214 is tolerant to various heavy metals, such as uranium, nickel, cobalt, and cadmium. We present its draft genome sequence here. PMID:23792748

  4. Draft Genome Sequences of Two Ralstonia pickettii Strains with Different Aminoglycoside Resistance Phenotypes

    PubMed Central

    Vaz-Moreira, Ivone; Martínez, José Luis

    2016-01-01

    The genomes of two Ralstonia pickettii strains (H2Cu2 and H2Cu5), isolated from hospital effluent in a selective medium containing CuSO4, were sequenced. They presented MICs of >256 and 6 µg/ml for the aminoglycoside gentamicin, respectively. The 5.2-Mb draft genomes have 40 contigs for strain H2Cu2 and 113 for H2Cu5. PMID:27834709

  5. Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains.

    PubMed

    Hasan, Syed A; Wietzes, Piet; Janssen, Dick B

    2012-02-01

    Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane-Andrew and Luong-Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10-15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.

  6. Inventorying the molecular potential of Cupriavidus and Ralstonia strains surviving harsh space-related environments

    NASA Astrophysics Data System (ADS)

    Mijnendonckx, Kristel; van Houdt, Rob; Provoost, Ann; Bossus, Albert; Ott, C. Mark; Venkateswaran, Kasthuri; Leys, Natalie

    The craving of modern man to explore life beyond earth presents a lot of challenges. The control of microbial contamination of the confined manned spacecraft is an important aspect that has to be taken into account in this journey. Because the human body contains a huge amount of microorganisms, the crew itself is the most important contamination source. But contamination can also originate from residing environmental microorganisms or from materials that are supplied from the Earth. These microbial contaminations can cause problems for the astronauts -well documented to have a decreased immunity -and the infrastructure of the space station. In this study, 14 different Cupriavidus metallidurans and Ralstonia pickettii strains, isolated from such space-related environments, where characterised in detail. These unique strains were isolated from drinking water that returned from ISS (3), from the cooling water system of the American ISS segment (4), from a swab sample of the Mars Odyssey Orbitor surface prior to flight (4), and from an air sample taken in the space assembly facility PHSF during Mars exploration Rover assembly (3). Their resistance to heavy metals and antibiotics was screened. The C. metallidurans isolates were more resistant to Zn2+ and Hg+ but more sensitive to Ni2+ than the R. pickettii strains. The MIC values for Cu2+ ranged from 1,5mM to 12mM, for Co2+ from 1,58mM to 12,63mM and for Cd2+ from 0,25mM to 1mM. For Ni2+ , the MIC values were between 2 and 8mM, except for the strain C. metallidurans IV (0502478) that was able to grow on Ni+2 concentrations up to 48mM. A metal of special interest was Ag+ because it is used to sanitize ISS drinking water. The strains isolated from air and surface samples showed a MIC value ranging from 0,35µM to 4µM. The isolates from the water samples had MIC values from 0,3µM to 2µM, which is lower than (or comparable with) the lowest limit of the silver concentration used in the ISS (1,9µM -4,6µM). However, all

  7. Pepper CabZIP63 acts as a positive regulator during Ralstonia solanacearum or high temperature-high humidity challenge in a positive feedback loop with CaWRKY40.

    PubMed

    Shen, Lei; Liu, Zhiqin; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Lin, Wei; Wang, Rongzhang; Yu, Huanxin; Mou, Shaoliang; Hussain, Ansar; Cheng, Wei; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-04-01

    CaWRKY40 is known to act as a positive regulator in the response of pepper (Capsicum annuum) to Ralstonia solanacearum inoculation (RSI) or high temperature-high humidity (HTHH), but the underlying mechanism remains elusive. Herein, we report that CabZIP63, a pepper bZIP family member, participates in this process by regulating the expression of CaWRKY40. CabZIP63 was found to localize in the nuclei, be up-regulated by RSI or HTHH, bind to promoters of both CabZIP63(pCabZIP63) and CaWRKY40(pCaWRKY40), and activate pCabZIP63- and pCaWRKY40-driven β-glucuronidase expression in a C- or G-box-dependent manner. Silencing of CabZIP63 by virus-induced gene silencing (VIGS) in pepper plants significantly attenuated their resistance to RSI and tolerance to HTHH, accompanied by down-regulation of immunity- or thermotolerance-associated CaPR1, CaNPR1, CaDEF1, and CaHSP24. Hypersensitive response-mediated cell death and expression of the tested immunity- and thermotolerance-associated marker genes were induced by transient overexpression (TOE) of CabZIP63, but decreased by that of CabZIP63-SRDX. Additionally, binding of CabZIP63 to pCaWRKY40 was up-regulated by RSI or HTHH, and the transcript level of CaWRKY40 and binding of CaWRKY40 to the promoters of CaPR1, CaNPR1, CaDEF1 and CaHSP24 were up-regulated by TOE of CabZIP63. On the other hand, CabZIP63 was also up-regulated transcriptionally by TOE of CaWRKY40. The data suggest collectively that CabZIP63 directly or indirectly regulates the expression of CaWRKY40 at both the transcriptional and post-transcriptional level, forming a positive feedback loop with CaWRKY40 during pepper's response to RSI or HTHH. Altogether, our data will help to elucidate the underlying mechanism of crosstalk between pepper's response to RSI and HTHH.

  8. Pepper CabZIP63 acts as a positive regulator during Ralstonia solanacearum or high temperature–high humidity challenge in a positive feedback loop with CaWRKY40

    PubMed Central

    Shen, Lei; Liu, Zhiqin; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Lin, Wei; Wang, Rongzhang; Yu, Huanxin; Mou, Shaoliang; Hussain, Ansar; Cheng, Wei; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-01-01

    CaWRKY40 is known to act as a positive regulator in the response of pepper (Capsicum annuum) to Ralstonia solanacearum inoculation (RSI) or high temperature–high humidity (HTHH), but the underlying mechanism remains elusive. Herein, we report that CabZIP63, a pepper bZIP family member, participates in this process by regulating the expression of CaWRKY40. CabZIP63 was found to localize in the nuclei, be up-regulated by RSI or HTHH, bind to promoters of both CabZIP63 (pCabZIP63) and CaWRKY40 (pCaWRKY40), and activate pCabZIP63- and pCaWRKY40-driven β-glucuronidase expression in a C- or G-box-dependent manner. Silencing of CabZIP63 by virus-induced gene silencing (VIGS) in pepper plants significantly attenuated their resistance to RSI and tolerance to HTHH, accompanied by down-regulation of immunity- or thermotolerance-associated CaPR1, CaNPR1, CaDEF1, and CaHSP24. Hypersensitive response-mediated cell death and expression of the tested immunity- and thermotolerance-associated marker genes were induced by transient overexpression (TOE) of CabZIP63, but decreased by that of CabZIP63-SRDX. Additionally, binding of CabZIP63 to pCaWRKY40 was up-regulated by RSI or HTHH, and the transcript level of CaWRKY40 and binding of CaWRKY40 to the promoters of CaPR1, CaNPR1, CaDEF1 and CaHSP24 were up-regulated by TOE of CabZIP63. On the other hand, CabZIP63 was also up-regulated transcriptionally by TOE of CaWRKY40. The data suggest collectively that CabZIP63 directly or indirectly regulates the expression of CaWRKY40 at both the transcriptional and post-transcriptional level, forming a positive feedback loop with CaWRKY40 during pepper’s response to RSI or HTHH. Altogether, our data will help to elucidate the underlying mechanism of crosstalk between pepper’s response to RSI and HTHH. PMID:26936828

  9. Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov.

    PubMed

    Vaneechoutte, Mario; Kämpfer, Peter; De Baere, Thierry; Falsen, Enevold; Verschraegen, Gerda

    2004-03-01

    Comparative 16S rDNA sequence analysis indicates that two distinct sublineages, with a sequence dissimilarity of >4 % (bootstrap value, 100 %), exist within the genus RALSTONIA: the Ralstonia eutropha lineage, which comprises Ralstonia basilensis, Ralstonia campinensis, R. eutropha, Ralstonia gilardii, Ralstonia metallidurans, Ralstonia oxalatica, Ralstonia paucula, Ralstonia respiraculi and Ralstonia taiwanensis; and the Ralstonia pickettii lineage, which comprises Ralstonia insidiosa, Ralstonia mannitolilytica, R. pickettii, Ralstonia solanacearum and Ralstonia syzygii comb. nov. (previously Pseudomonas syzygii). This phylogenetic discrimination is supported by phenotypic differences. Members of the R. eutropha lineage have peritrichous flagella, do not produce acids from glucose and are susceptible to colistin, in contrast to members of the R. pickettii lineage, which have one or more polar flagella, produce acid from several carbohydrates and are colistin-resistant. Members of the R. pickettii lineage are viable for up to 6 days on tryptic soy agar at 25 degrees C, whereas members of the R. eutropha lineage are viable for longer than 9 days. It is proposed that species of the R. eutropha lineage should be classified in a novel genus, Wautersia gen. nov. Finally, based on the literature and new DNA-DNA hybridization data, it is proposed that Pseudomonas syzygii should be renamed Ralstonia syzygii comb. nov.

  10. Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB(-)4 as host strain.

    PubMed

    Hyakutake, Manami; Saito, Yuta; Tomizawa, Satoshi; Mizuno, Kouhei; Tsuge, Takeharu

    2011-01-01

    Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).

  11. Identification of the mcpA and mcpM genes, encoding methyl-accepting proteins involved in amino acid and l-malate chemotaxis, and involvement of McpM-mediated chemotaxis in plant infection by Ralstonia pseudosolanacearum (formerly Ralstonia solanacearum phylotypes I and III).

    PubMed

    Hida, Akiko; Oku, Shota; Kawasaki, Takeru; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

    2015-11-01

    Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.

  12. Identification of the mcpA and mcpM Genes, Encoding Methyl-Accepting Proteins Involved in Amino Acid and l-Malate Chemotaxis, and Involvement of McpM-Mediated Chemotaxis in Plant Infection by Ralstonia pseudosolanacearum (Formerly Ralstonia solanacearum Phylotypes I and III)

    PubMed Central

    Hida, Akiko; Oku, Shota; Kawasaki, Takeru; Tajima, Takahisa

    2015-01-01

    Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand. PMID:26276117

  13. Genome sequences of Ralstonia insidiosa type strain ATCC 49129 and strain FC1138, a strong biofilm producer isolated from a fresh-cut produce-processing plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia insidiosa FC1138 is a strong biofilm producer, isolated from a local fresh-cut produce processing plant. Here, we present the complete genome sequence of Ralstonia insidiosa FC1138 which includes two circular chromosomes and a plasmid. To our knowledge, this is the first reported complete ...

  14. Novel Gene Clusters and Metabolic Pathway Involved in 3,5,6-Trichloro-2-Pyridinol Degradation by Ralstonia sp. Strain T6

    PubMed Central

    Li, Jingquan; Huang, Yan; Hou, Ying; Li, Xiangmin; Cao, Hui

    2013-01-01

    3,5,6-Trichloro-2-pyridinol (TCP) is a widespread pollutant. Some bacteria and fungi have been reported to degrade TCP, but the gene clusters responsible for TCP biodegradation have not been characterized. In this study, a fragment of the reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase gene tcpA was amplified from the genomic DNA of Ralstonia sp. strain T6 with degenerate primers. The tcpA disruption mutant strain T6-ΔtcpA could not degrade TCP but could degrade the green intermediate metabolite 3,6-dihydroxypyridine-2,5-dione (DHPD), which was generated during TCP biodegradation by strain T6. The flanking sequences of tcpA were obtained by self-formed adaptor PCR. tcpRXA genes constitute a gene cluster. TcpR and TcpX are closely related to the LysR family transcriptional regulator and flavin reductase, respectively. T6-ΔtcpA-com, the complementation strain for the mutant strain T6-ΔtcpA, recovered the ability to degrade TCP, and the strain Escherichia coli DH10B-tcpRXA, which expressed the tcpRXA gene cluster, had the ability to transform TCP to DHPD, indicating that tcpA is a key gene in the initial step of TCP degradation and that TcpA dechlorinates TCP to DHPD. A library of DHPD degradation-deficient mutants of strain T6 was obtained by random transposon mutagenesis. The fragments flanking the Mariner transposon were amplified and sequenced, and the dhpRIJK gene cluster was cloned. DhpJ could transform DHPD to yield an intermediate product, 5-amino-2,4,5-trioxopentanoic acid (ATOPA), which was further degraded by DhpI. DhpR and DhpK are closely related to the AraC family transcriptional regulator and the MFS family transporter, respectively. PMID:24056464

  15. Genomic Characterization of the Filamentous Integrative Bacteriophages φRSS1 and φRSM1, Which Infect Ralstonia solanacearum▿

    PubMed Central

    Kawasaki, Takeru; Nagata, Shoko; Fujiwara, Akiko; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

    2007-01-01

    The genomic DNA sequences were determined for two filamentous integrative bacteriophages, φRSS1 and φRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of φRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within φRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the φRSS1 intergenic (IG) region. The 9,004-base sequence of φRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the φRSM1 replication module. Genomic DNA fragments containing the φRSM integration junctions were cloned and sequenced from φRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5′-TGGCGGAGAGGGT-3′, corresponding to the 3′ end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the φRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of φRSS1 is within a sequence element of the IG region. PMID:17557818

  16. Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

    PubMed Central

    Zhou, Ning-Yi; Al-Dulayymi, Jumáa; Baird, Mark S.; Williams, Peter A.

    2002-01-01

    The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the “classical” naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2. PMID:11872705

  17. Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

    PubMed Central

    Tumlirsch, Tony; Sznajder, Anna

    2015-01-01

    A protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion of phaX resulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs. In vivo colocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of the Ralstonia eutropha ppk (ppkReu) genes in an Escherichia coli Δppk background and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds. PMID:26407880

  18. The Naphthalene Catabolic (nag) Genes of Ralstonia sp. Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator

    PubMed Central

    Jones, Rheinallt M.; Britt-Compton, Bethan; Williams, Peter A.

    2003-01-01

    In Ralstonia sp. strain U2, the nag catabolic genes, which encode the enzymes for the pathway that catabolizes naphthalene via the alternative ring cleavage gentisate pathway, are transcribed as an operon under the same promoter. nagR, which encodes a LysR-type transcriptional regulator, is divergently transcribed compared to the nag catabolic genes. A 4-bp frameshift deletion in nagR demonstrated that NagR is required for expression of the nag operon. The transcriptional start of the nag operon was mapped, and a putative −10, −35 σ70-type promoter binding site was identified. Further upstream, a site proximal to the promoter was identified as a site that has bases which have been found to be conserved in the activator-binding motif of other naphthalene pathways. Transcriptional fusion studies demonstrated that NagR regulates the expression of the nag operon positively in the presence of salicylate and to a lesser extent in the presence of 2-nitrobenzoate. Mutation of the LysR-type activator-binding motif in the nag promoter-proximal region resulted in a loss of inducibility of a lacZ reporter gene transcriptionally fused to nagAa, the first gene of the operon. However, other mutations in the region increased the effectiveness of salicylate as an inducer. PMID:13129957

  19. Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

    PubMed Central

    Dionisi, Hebe M.; Chewning, Christopher S.; Morgan, Katherine H.; Menn, Fu-Min; Easter, James P.; Sayler, Gary S.

    2004-01-01

    We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) × 103 to (2.9 ± 0.3) × 105 copies of nagAc-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 105 to (5.4 ± 0.4) × 107 copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene. PMID:15240274

  20. Purification and gene cloning of alpha-methylserine aldolase from Ralstonia sp. strain AJ110405 and application of the enzyme in the synthesis of alpha-methyl-L-serine.

    PubMed

    Nozaki, Hiroyuki; Kuroda, Shinji; Watanabe, Kunihiko; Yokozeki, Kenzo

    2008-12-01

    By screening microorganisms that are capable of assimilating alpha-methyl-DL-serine, we detected alpha-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of alpha-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from alpha-methyl-L-serine but not from alpha-methyl-D-serine, L-serine, or D-serine. Alpha-methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the alpha-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding alpha-methylserine aldolase and effectively obtained a high yield of optically pure alpha-methyl-L-serine using L-alanine and formaldehyde.

  1. Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

    PubMed

    Lin, Kaichien; Elbahloul, Yasser; Steinbüchel, Alexander

    2012-03-01

    The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

  2. CDC Group IV c-2: a New Ralstonia Species Close to Ralstonia eutropha

    PubMed Central

    Moissenet, Didier; Goujon, Christophe P.; Garbarg-Chenon, Antoine; Vu-Thien, Hoang

    1999-01-01

    CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus Ralstonia, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus Ralstonia. The closest matches were obtained with Ralstonia eutropha. However, four differences in 32 biochemical tests separated R. eutropha from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus Ralstonia. PMID:10325323

  3. New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations.

    PubMed

    Guinard, Jérémy; Latreille, Anne; Guérin, Fabien; Poussier, Stéphane; Wicker, Emmanuel

    2017-03-01

    Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations.IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the

  4. Characterization and evaluation of Bacillus amyloliquefaciens strain WF02 regarding its biocontrol activities and genetic responses against bacterial wilt in two different resistant tomato cultivars.

    PubMed

    Huang, Chu-Ning; Lin, Chan-Pin; Hsieh, Feng-Chia; Lee, Sook-Kuan; Cheng, Kuan-Chen; Liu, Chi-Te

    2016-11-01

    Bacillus amyloliquefaciens strain WF02, isolated from soil collected at Wufeng Mountain, Taiwan, has siderophore-producing ability and in vitro antagonistic activity against bacterial wilt pathogen. To determine the impact of plant genotype on biocontrol effectiveness, we treated soil with this strain before infecting susceptible (L390) and moderately resistant (Micro-Tom) tomato cultivars with Ralstonia solanacearum strain Pss4. We also compared the efficacy of this strain with that of commercial Bacillus subtilis strain Y1336. Strain WF02 provided longer lasting protection against R. solanacearum than did strain Y1336 and controlled the development of wilt in both cultivars. To elucidate the genetic responses in these plants under WF02 treatment, we analyzed the temporal expression of defense-related genes in leaves. The salicylic acid pathway-related genes phenylalanine ammonia-lyase and pathogenesis-related protein 1a were up-regulated in both cultivars, whereas expression of the jasmonic acid pathway-related gene lipoxygenase was only elevated in the susceptible tomato cultivar (L390). These results suggest that WF02 can provide protection against bacterial wilt in tomato cultivars with different levels of disease resistance via direct and indirect modes of action.

  5. Identification and Chacterization of new strains of Enterobacter spp. causing Mulberry (Morus alba) wilt disease in China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are dark brown discolorations in vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the bacterial wilt disease caused by Ralstonia solanacearum, the leaf w...

  6. Protein engineering of the archetypal nitroarene dioxygenase of Ralstonia sp. strain U2 for activity on aminonitrotoluenes and dinitrotoluenes through alpha-subunit residues leucine 225, phenylalanine 350, and glycine 407.

    PubMed

    Keenan, Brendan G; Leungsakul, Thammajun; Smets, Barth F; Mori, Masa-aki; Henderson, David E; Wood, Thomas K

    2005-05-01

    Naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the alpha-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases.

  7. Protein Engineering of the Archetypal Nitroarene Dioxygenase of Ralstonia sp. Strain U2 for Activity on Aminonitrotoluenes and Dinitrotoluenes through Alpha-Subunit Residues Leucine 225, Phenylalanine 350, and Glycine 407

    PubMed Central

    Keenan, Brendan G.; Leungsakul, Thammajun; Smets, Barth F.; Mori, Masa-aki; Henderson, David E.; Wood, Thomas K.

    2005-01-01

    Naphthalene dioxygenase (NDO) from Ralstonia sp. strain U2 has not been reported to oxidize nitroaromatic compounds. Here, saturation mutagenesis of NDO at position F350 of the α-subunit (NagAc) created variant F350T that produced 3-methyl-4-nitrocatechol from 2,6-dinitrotoluene (26DNT), that released nitrite from 23DNT sixfold faster than wild-type NDO, and that produced 3-amino-4-methyl-5-nitrocatechol and 2-amino-4,6-dinitrobenzyl alcohol from 2-amino-4,6-dinitrotoluene (2A46DNT) (wild-type NDO has no detectable activity on 26DNT and 2A46DNT). DNA shuffling identified the beneficial NagAc mutation G407S, which when combined with the F350T substitution, increased the rate of NDO oxidation of 26DNT, 23DNT, and 2A46DNT threefold relative to variant F350T. DNA shuffling of NDO nagAcAd also generated the NagAc variant G50S/L225R/A269T with an increased rate of 4-amino-2-nitrotoluene (4A2NT; reduction product of 2,4-dinitrotoluene) oxidation; from 4A2NT, this variant produced both the previously uncharacterized oxidation product 4-amino-2-nitrocresol (enhanced 11-fold relative to wild-type NDO) as well as 4-amino-2-nitrobenzyl alcohol (4A2NBA; wild-type NDO does not generate this product). G50S/L225R/A269T also had increased nitrite release from 23DNT (14-fold relative to wild-type NDO) and generated 2,3-dinitrobenzyl alcohol (23DNBA) fourfold relative to wild-type NDO. The importance of position L225 for catalysis was confirmed through saturation mutagenesis; relative to wild-type NDO, NDO variant L225R had 12-fold faster generation of 4-amino-2-nitrocresol and production of 4A2NBA from 4A2NT as well as 24-fold faster generation of nitrite and 15-fold faster generation of 23DNBA from 23DNT. Hence, random mutagenesis discovered two new residues, G407 and L225, that influence the regiospecificity of Rieske non-heme-iron dioxygenases. PMID:15866914

  8. First report of bacterial wilt caused by Ralstonia solanacearum on Mesona chinensis in China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Jellywort (Mesona chinensis Benth) is a herbaceous plant in the Lamiaceae Family. The plant is referred to as ‘Xiancao’ (Weed from Angels) in Chinese and is primarily used to make grass jelly, a popular refreshing drink. Currently, Xiancao cultivation is a fast growing industry with a high profit ma...

  9. Enzymatic modification of chitosan by cinnamic acids: Antibacterial activity against Ralstonia solanacearum.

    PubMed

    Yang, Caifeng; Zhou, Yu; Zheng, Yu; Li, Changlong; Sheng, Sheng; Wang, Jun; Wu, Fuan

    2016-06-01

    This study aimed to identify chitosan polymers that have antibacterial activity against the bacterial wilt pathogen. The chitosan polymers were enzymatically synthesized using chitosan and five cinnamic acids (CADs): caffeic acid (CA), ferulic acid (FA), cinnamic acid (CIA), p-coumaric acid (COA) and chlorogenic acid (CHA), using laccase from Pleurotus ostreatus as a catalyst. The reaction was performed in a phosphate buffered solution under heterogenous reaction conditions. The chitosan derivatives (CTS-g-CADs) were characterized by FT-IR, XRD, TGA and SEM. FT-IR demonstrated that the reaction products bound covalently to the free amino groups or hydroxyl groups of chitosan via band of amide I or ester band. XRD showed a reduced packing density for grafted chitosan comparing to original chitosan. TGA demonstrated that CTS-g-CADs have a higher thermostability than chitosan. Additionally, chitosan and its derivatives showed similar antibacterial activity. However, the IC50 value of the chitosan-caffeic acid derivative (CTS-g-CA) against the mulberry bacterial wilt pathogen RS-5 was 0.23mg/mL, which was two-fifths of the IC50 value of chitosan. Therefore, the enzymatically synthesized chitosan polymers can be used to control plant diseases in biotechnological domains.

  10. Novel Tn4371-ICE like element in Ralstonia pickettii and Genome mining for comparative elements

    PubMed Central

    2009-01-01

    Background Integrative Conjugative Elements (ICEs) are important factors in the plasticity of microbial genomes. An element related to the ICE Tn4371 was discovered during a bioinformatic search of the Ralstonia pickettii 12J genome. This element was analysed and further searches carried out for additional elements. A PCR method was designed to detect and characterise new elements of this type based on this scaffold and a culture collection of fifty-eight Ralstonia pickettii and Ralstonia insidiosa strains were analysed for the presence of the element. Results Comparative sequence analysis of bacterial genomes has revealed the presence of a number of uncharacterised Tn4371-like ICEs in the genomes of several β and γ- Proteobacteria. These elements vary in size, GC content, putative function and have a mosaic-like structure of plasmid- and phage-like sequences which is typical of Tn4371-like ICEs. These elements were found after a through search of the GenBank database. The elements, which are found in Ralstonia, Delftia, Acidovorax, Bordetella, Comamonas, Acidovorax, Congregibacter, Shewanella, Pseudomonas Stenotrophomonas, Thioalkalivibrio sp. HL-EbGR7, Polaromonas, Burkholderia and Diaphorobacter sp. share a common scaffold. A PCR method was designed (based on the Tn4371- like element detected in the Ralstonia pickettii 12J genome) to detect and characterise new elements of this type. Conclusion All elements found in this study possess a common scaffold of core genes but contain different accessory genes. A new uniform nomenclature is suggested for ICEs of the Tn4371 family. Two novel Tn4371-like ICE were discovered and characterised, using the novel PCR method described in two different isolates of Ralstonia pickettii from laboratory purified water. PMID:19941653

  11. Hrp- Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

    PubMed Central

    Frey, Pascal; Prior, Philippe; Marie, Corinne; Kotoujansky, Alain; Trigalet-Demery, Daniele; Trigalet, Andre

    1994-01-01

    There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp- mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ω-Km interposon within the hrp gene cluster of each strain. The resulting Hrp- mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp- mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants. Images PMID:16349373

  12. Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov.

    PubMed

    Coenye, T; Falsen, E; Vancanneyt, M; Hoste, B; Govan, J R; Kersters, K; Vandamme, P

    1999-04-01

    A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis, whole-cell protein and fatty acid analyses, AFLP (amplified fragment length polymorphism) fingerprinting and an extensive biochemical characterization was performed on 10 strains provisionally identified as Alcaligenes faecalis-like bacteria. The six environmental and four human isolates belonged to the genus Ralstonia and were assigned to a new species for which the name Ralstonia gilardii sp. nov. is proposed. The type strain is LMG 5886T.

  13. Draft Genome Sequence of Ralstonia sp. MD27, a Poly(3-Hydroxybutyrate)-Degrading Bacterium, Isolated from Compost

    PubMed Central

    Zhu, Morgan; McCully, Lucy M.; Silby, Mark W.; Charles-Ogan, Tamunonengiyeofori I.

    2015-01-01

    Ralstonia sp. strain MD27, a novel biopolymer-degrading betaproteobacterium, was isolated from compost samples. This organism has been shown to utilize the biopolymer poly(3-hydroxybutyrate) [P(3HB)] as a carbon source for growth. We report the draft genome sequence of MD27 with an estimated total sequence length of 5.9 Mb. PMID:26450738

  14. Ralstonia insidiosa serves as bridges in biofilm formation by foodborne pathogens Listeria monocytogenes, Salmonella enterica, and enterohemorrhagic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm formation on abiotic surfaces in fresh produce processing facilities might play a role in foodborne outbreaks by providing protective microniches for pathogenic bacteria. Our previous study showed that a strain of Ralstonia insidiosa isolated from a fresh produce processing plant could enhan...

  15. Effect of organic fertilizers prepared from organic waste materials on the production of antibacterial volatile organic compounds by two biocontrol Bacillus amyloliquefaciens strains.

    PubMed

    Raza, Waseem; Wei, Zhong; Ling, Ning; Huang, Qiwei; Shen, Qirong

    2016-06-10

    Three organic fertilizers made of different animal and plant waste materials (BOFs) were evaluated for their effects on the production of antibacterial volatile organic compounds (VOCs) by two Bacillus amyloliquefaciens strains SQR-9 and T-5 against the tomato wilt pathogen Ralstonia solanacearum (RS). Both strains could produce VOCs that inhibited the growth and virulence traits of RS; however, in the presence of BOFs, the production of antibacterial VOCs was significantly increased. The maximum inhibition of growth and virulence traits of RS by VOCs of T-5 and SQR-9 was determined at 1.5% BOF2 and 2% BOF3, respectively. In case of strain T-5, 2-nonanone, nonanal, xylene, benzothiazole, and butylated hydroxy toluene and in case of strain SQR-9, 2-nonanone, nonanal, xylene and 2-undecanone were the main antibacterial VOCs whose production was increased in the presence of BOFs. The results of this study reveal another significance of using organic fertilizers to improve the antagonistic activity of biocontrol agents against phytopathogens.

  16. Genomic Plasticity in Ralstonia eutropha and Ralstonia pickettii: Evidence for Rapid Genomic Change and Adaptation

    SciTech Connect

    Terence L. MArsh

    2007-06-27

    The proposed foci of our investigations were on Ralstonia eutropha and Rasltonia pickettii. We have 18 derived lineages of the former as well as their progenitor and eleven isolates of the latter. Our goal was to measure the level of plasticity in these strains and attempt to derive a mechanistic understanding of how genomic plasticity formed. Extensive attempts to reproducibly induce conformational changes in the genome of R. eutropha were unsuccessful. We thought that we had a reasonable lead on this inasmuch as we had shown that the ancestral strain along with many of the derivative lineages exhibited “temperature induced mutation and mortality akin to R. metallodurans. However we were unable to get subtractive hybridization working to the degree that it revealed differences between the lineages. During this time the R. pickettii analysis was proving quite fruitful and so we concentrated our efforts on our analyses of R. pickettii. These strains were isolated from a copper-contaminated lake sediment and were resistant to copper at 800 µg/ml (CuSO4). Our results in the investigation of R. pickettii permitted a view into the adaptation of a beta-proteobacteria to an extreme environment. Our worked revealed that within the same ecosystem two genomovars with structurally different genomes and genome sizes were present and apparently filling similar if not identical niches. The genomovars were detected with REP & BOX-PCR, pulse field gel electrophoresis, and DNA:DNA hybridizations. Moreover there were different metal resistance patterns associated with the different genomovars, one showing resistance to Zn and Cd while the other had resistance to Ni. Five of the isolates had a high-copy number extrachromosomal element that was identified as the replicative form of a filamentous phage. Mature virions were isolated from culture broth using PEG precipitation and CsCl density centrifugation. The DNA associated with the filamentous particles was single stranded and had

  17. Isolation and antifungal activity of 4-phenyl-3-butenoic acid from Streptomyces koyangensis strain VK-A60.

    PubMed

    Lee, Jee Yeon; Lee, Jung Yeop; Moon, Surk Sik; Hwang, Byung Kook

    2005-10-05

    An antifungal compound was isolated from the culture broth of Streptomyces koyangensis strain VK-A60 using various chromatographic procedures. On the basis of the high-resolution EI-mass and 1H and 13C NMR data, the compound was identified as 4-phenyl-3-butenoic acid. Colletotrichum orbiculare, Magnaporthe grisea, and Pythium ultimum were most sensitive to 4-phenyl-3-butenoic acid. Strong inhibitory effects of 4-phenyl-3-butenoic acid also were found against Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum. 4-Phenyl-3-butenoic acid effectively suppressed the development of M. grisea on rice leaves at the concentration of more than 10 microg/mL, and the protective activity was in general similar to that of the commercial fungicide tricyclazole. Treatment with 100 microg/mL of 4-phenyl-3-butenoic acid also effectively inhibited the anthracnose development on cucumber plants, although its in vivo efficacy was somewhat less effective than that of the commercial fungicide chlorothalonil.

  18. Incorporation of Escherichia coli O157:H7 in dual-species biofilms with Ralstonia insidiosa, a primary colonizer for the development of heterogeneous biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of strong biofilm forming microflora could potentially enhance the survival of Escherichia coli O157:H7 (EcO157) in harsh environment. One strain of Ralstonia insidiosa isolated from produce processing environments, previously displayed a synergistic interaction with EcO157 in dual-spec...

  19. Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli

    PubMed Central

    Drewlo, Sascha; Brämer, Christian O.; Madkour, Mohamed; Mayer, Frank; Steinbüchel, Alexander

    2001-01-01

    On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo. PMID:11282658

  20. Ralstonia mannitolilytica-Induced Septicemia and Homology Analysis in Infected Patients: 3 Case Reports

    PubMed Central

    Liu, Cai-Xia; Yan, Chun; Zhang, Pan; Li, Fang-Qu; Yang, Jing-Hong; Li, Xiang-Yang

    2016-01-01

    Background Ralstonia mannitolilytica is an emerging opportunistic pathogen. Hospital outbreaks of Ralstonia spp. are mainly associated with contaminated treatment water or auxiliary instruments. Objectives In this report, we summarize the clinical infection characteristics of R. mannitolilytica, the drug-susceptibility testing of the bacterial strains, and the results of related infection investigations. Patients and Methods We retrospectively analyzed the clinical information of 3 patients with R. mannitolilytica. Results The patients’ primary-onset symptoms were chills and fever. The disease progressed rapidly and septic shock symptoms developed. Laboratory tests indicated progressively decreased white blood cells and platelets, as well as significant increases in certain inflammation indicators. The effect of treatment with Tazocin was good. The growth period of R. mannitolilytica in sterile distilled water was > 6 months. The pulsed-field gel electrophoresis (PFGE) results revealed that the infectious strains from these 3 patients were not the same clonal strain. This bacterium was not detected in the nosocomial infection samples. Conclusions Our results suggest that R. mannitolilytica-induced septicemia had an acute disease onset and rapid progression. The preferred empirical antibiotic was Tazocin. In these 3 cases, the R. mannitolilytica-induced septicemia was not due to clonal transmission. PMID:27679705

  1. Cleaning-resistant Cupriavidus and Ralstonia bacteria contaminating spacecrafts and the ultra clean rooms they are assembled in.

    NASA Astrophysics Data System (ADS)

    Leys, N.; Dams, A.; Bossus, A.; Provoost, A.; Venkateswaran, K.; Mergeay, M.

    Background Planetary Protection is preventing microbial contamination of both the target planet and the Earth when sending spacecrafts on interplanetary space mission It is important to preserve the natural conditions of other planets and to not bring with robots earthly microbes forward contamination when looking for spores of extra terrestrial life Spacecrafts and the ultra clean rooms they are assembled in are routinely monitored for microbial contamination It was shown that the floor air and surfaces of such spacecraft assembly rooms often contain Cupriavidu s and Ralstonia bacteria These bacteria not only contaminated the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter La Duc et al 2003 and even in-flight in ISS cooling water and Shuttle drinking water unpublished Aim In this study several Cupriavidus and Ralstonia strains isolated from space craft assembling rooms and spacecrafts were characterized and analysed in detail Results The analysis showed that all the Cupriavidus and Ralstonia clean-room isolates are able to use a wide variety of substrates as carbon sources including ethanol and acetone In addition they all have accumulated moderate resistances to an extraordinary collection of physical and chemical antimicrobial agents Some of the test strains were able to form biofilms on plastic and metal materials used for space robots a nutritional and

  2. Sequence analysis of Candidatus Liberibacter solanacearum (Lso-C) isolated from carrot psyllids collected in Scandinavia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fastidious prokaryote Candidatus Liberibacter solanacearum (Lso), transmitted by the tomato potato psyllid (Bactericera cockerelli), is associated with the Zebra Chip disease of potato. Plants infected with Liberibacter may experience significant yield losses and these plants also serve as pote...

  3. First Report of 'Candidatus Liberibacter Solanacearum' Naturally Infecting Tomatoes in the State of Mexico, Mexico

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato (Solanum lycopersicum) plants exhibiting stunting, yellow mosaic, short, chlorotic leaves, aborted flowers and reduced-size fruits, symptoms similar to those exhibited by plants infected by Candidatus Liberibacter solanacearum (Lso), were observed in greenhouses in Jocotitlan, Mexico. In addi...

  4. Effect of selected environmental factors on degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost.

    PubMed

    Hundt, K; Wagner, M; Becher, D; Hammer, E; Schauer, F

    1998-04-01

    By varying selected environmental factors, the degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost were investigated. An optimized soil moisture and enhanced bioavailability by using the nonionic surfactant Tween 80 were of great importance for the degradation rates of biaryl compounds like biphenyl and 4-chlorobiphenyl by cells of Ralstonia picketti SBUG 290 inoculated into soil. Additionally, degradation of these compounds by the investigated strain in soil was strongly dependent upon the medium of precultivation. Also the influence of temperature and soil pH-value was tested. In contrast to the used soil, the autochthonous flora of the compost seemed to have a higher physiological activity. All investigated compounds (biphenyl, 4-chlorobiphenyl and dibenzofuran) were degraded quickly in compost. Inoculation with the investigated bacterium did not enhance the degradation rates significantly.

  5. Native Valve Endocarditis due to Ralstonia pickettii: A Case Report and Literature Review

    PubMed Central

    Orme, Joseph; Rivera-Bonilla, Tomas; Loli, Akil; Blattman, Negin N.

    2015-01-01

    Ralstonia pickettii is a rare pathogen and even more rare in healthy individuals. Here we report a case of R. pickettii bacteremia leading to aortic valve abscess and complete heart block. To our knowledge this is the first case report of Ralstonia species causing infective endocarditis with perivalvular abscess. PMID:25648998

  6. Native Valve Endocarditis due to Ralstonia pickettii: A Case Report and Literature Review.

    PubMed

    Orme, Joseph; Rivera-Bonilla, Tomas; Loli, Akil; Blattman, Negin N

    2015-01-01

    Ralstonia pickettii is a rare pathogen and even more rare in healthy individuals. Here we report a case of R. pickettii bacteremia leading to aortic valve abscess and complete heart block. To our knowledge this is the first case report of Ralstonia species causing infective endocarditis with perivalvular abscess.

  7. EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance.

    PubMed

    Créchet, Jean-Bernard; Malosse, Christian; Hountondji, Codjo

    2016-08-01

    In this report, we have demonstrated that the poly(U)-dependent poly(Phe) synthesis activity of elongator factor Tu (EF-Tu) from the enacyloxin producing strain Frateuria sp. W-315 is inhibited by the antibiotic similarly to that of Escherichia coli EF-Tu. The inhibitory effect of enacyloxin observed in a purified system was the same as that obtained with an S30 extract from E. coli or Frateuria sp. W-315, respectively, suggesting that antibiotic resistance of enacyloxin producing Frateuria sp. W-315 is not due neither to EF-Tu nor to other components of the translation machinery but to a still unknown mechanism. The EF-Tu gene, as PCR amplified from Frateuria W-315 genomic DNA and sequenced represented an ORF of 1191 nucleotides corresponding to 396 amino acids. This protein is larger than the product of tufA from E. coli by only two amino acid residues. Alignment of the amino acid sequence of EF-Tu from E. coli with those of Frateuria and Ralstonia solanacearum indicates on average 80% identical amino acid residues and 9.7% conservative replacements between EF-Tu Frateuria and EF-Tu E. coli, on one hand, and 97% identity and 1.7% conservative replacement between EF-Tu Frateuria and EF-Tu Ralstonia solanacearum, on the other hand. These strong primary structure similarities between EF-Tu from different origins are consistent with the fact that this factor is essential for the translation process in all kingdoms of life. Comparison of the effects of antibiotics on EF-Tu Frateuria and EF-Tu E. coli revealed that enacyloxin, kirromycin and pulvomycin exert a stronger stimulation of the GDP dissociation rate on EF-Tu Frateuria, while the effects of the antibiotics on the GDP association rate were comparable for the two EF-Tu species. Different mutants of EF-Tu E. coli were constructed with the help of site directed mutagenesis by changing one or several residues of EF-Tu E. coli by the corresponding residues of EF-Tu Frateuria. The single A45K substitution did

  8. Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates.

    PubMed

    Park, Si Jae; Jang, Young-Ah; Lee, Hyuk; Park, A-Reum; Yang, Jung Eun; Shin, Jihoon; Oh, Young Hoon; Song, Bong Keun; Jegal, Jonggeon; Lee, Seung Hwan; Lee, Sang Yup

    2013-11-01

    Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6-19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.

  9. Engineering of Ralstonia eutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose.

    PubMed

    Zhang, Ying-Zi; Liu, Gui-Ming; Weng, Wei-Qi; Ding, Jiu-Yuan; Liu, Shuang-Jiang

    2015-02-10

    Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on the addition of propionate during fermentation, and propionate consumption is one of the major factors affecting the cost of PHBV production. In this study, 7 strains were obtained by genetic manipulating the methylcitric acid cycle and the methylmalonyl-CoA pathway in R. eutropha. Disruption of prpC1 and prpC2 genes did not affect cell growth and PHBV accumulation. All 7 strains were able to accumulation high amounts of PHBVs with 3HV fractions of 0.41-29.1 mol% during cultivation in flasks. Fermentation in 7.5-L fermenter showed that genetically engineered Rem-8 was able to yield biomass of 132.8 CDWg/L, of which 68.6% were PHBV with 3HV fraction of 26.0 mol% in the biopolymer, indicating promising potentials of commercialization in the future.

  10. Suppression of bacterial wilt of tomato by bioorganic fertilizer made from the antibacterial compound producing strain Bacillus amyloliquefaciens HR62.

    PubMed

    Huang, Jianfeng; Wei, Zhong; Tan, Shiyong; Mei, Xinlan; Shen, Qirong; Xu, Yangchun

    2014-11-05

    Ralstonia solanacearum (Smith) is an important soil-borne pathogen worldwide. We investigated the effects of a new bioorganic fertilizer, BIO62, which was made from organic fertilizer and antagonist Bacillus amyloliquefaciens HR62, on the control of bacterial wilt of tomato in greenhouse condition. The results showed that the application of BIO62 significantly decreased disease incidence by 65% and strongly reduced R. solanacearum populations both in the rhizosphere soil (8.04 log cfu g(-1) dry soil) and crown sections (5.63 log cfu g(-1) fresh plant section) at 28 days after pathogen challenge. Antibacterial compounds produced by HR62 were purified by silica gel, Sephadex LH-20, and HPLC and then identified using HPLC/electrospray ionization mass spectrometry analysis. Macrolactin A and 7-O-malonyl macrolactin A (molecular weights of 402 and 488 Da, respectively), along with surfactin B (molecular weights of 994, 1008, 1022, and 1036 Da), were observed to inhibit the growth of R. solanacearum.

  11. Ralstonia pickettii-Induced Ataxia in Immunodeficient Mice

    PubMed Central

    Berard, Marion; Medaille, Christine; Simon, Meredith; Serre, Stéphanie; Pritchett-Corning, Kathleen; Dangles-Marie, Virginie

    2009-01-01

    We report here the characterization of an asymmetric ataxia syndrome (head tilt and circling, with death in the most severe cases) demonstrated by profoundly immunodeficient mice housed at the Institut Curie SPF facility. The immune system of the affected mice had been genetically modified so that they were deficient in both B and T cells. Extensive bacteriologic, parasitic, serologic, and histopathologic analysis of the affected animals and their healthy controls led us to identify Ralstonia pickettii as the causative agent of the ataxic syndrome. The outbreak was managed through a test-and-cull process. Even though they also carried Ralstonia pickettii, immunocompetent mice that were kept in the same facility, did not show any of the signs that were expressed by their immunodeficient counterparts. This case highlights the difficulty of maintaining immunocompetent and immunodeficient mice in the same microbiologic unit and the importance of enlarging the spectrum of health monitoring to opportunistic agents when investigating clinical cases in populations of immunocompromised rodents. PMID:19389312

  12. Phenotypic evaluation of the Chinese mini-mini core collection of peanut (Arachis hypogaea L.) and assessment for resistance to bacterial wilt disease caused by Ralstonia solanacearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to utilize the germplasm more efficiently for peanut (Arachis hypogaea L.) genetic improvement, a core collection of 576 accessions and a primary mini core collection of 298 accessions was developed previously from a collection of 6,839 cultivated peanut lines stored at the Oil Crops Resear...

  13. Production of branched-chain alcohols by recombinant Ralstonia eutropha in fed-batch cultivation

    SciTech Connect

    Fei, Q; Brigham, CJ; Lu, JN; Fu, RZ; Sinskey, AJ

    2013-09-01

    Branched-chain alcohols are considered promising green energy sources due to their compatibility with existing infrastructure and their high energy density. We utilized a strain of Ralstonia eutropha capable of producing branched-chain alcohols and examined its production in flask cultures. In order to increase isobutanol and 3-methyl-1-butanol (isoamyl alcohol) productivity in the engineered strain, batch, fed-batch, and two-stage fed-batch cultures were carried out in this work. The effects of nitrogen source concentration on branched-chain alcohol production were investigated under four different initial concentrations in fermenters. A maximum 380 g m(-3) of branched-chain alcohol production was observed with 2 kg m(-3) initial NH4Cl concentration in batch cultures. A pH-stat control strategy was utilized to investigate the optimum carbon source amount fed during fed-batch cultures for higher cell density. In cultures of R. eutropha strains that did not produce polyhydroxyalkanoate or branched-chain alcohols, a maximum cell dry weight of 36 kg m(-3) was observed using a fed-batch strategy, when 10 kg m(-3) carbon source was fed into culture medium. Finally, a total branched-chain alcohol titer of 790 g m(-3), the highest branched-chain alcohol yield of 0.03 g g(-1), and the maximum branched-chain alcohol productivity of 8.23 g m(-3) h(-1) were obtained from the engineered strain Re2410/pJL26 in a two-stage fed-batch culture system with pH-stat control. Isobutanol made up over 95% (mass fraction) of the total branched-chain alcohols titer produced in this study. (C) 2013 Published by Elsevier Ltd.

  14. Engineering the heterotrophic carbon sources utilization range of Ralstonia eutropha H16 for applications in biotechnology.

    PubMed

    Volodina, Elena; Raberg, Matthias; Steinbüchel, Alexander

    2016-12-01

    Ralstonia eutropha H16 is an interesting candidate for the biotechnological production of polyesters consisting of hydroxy- and mercaptoalkanoates, and other compounds. It provides all the necessary characteristics, which are required for a biotechnological production strain. Due to its metabolic versatility, it can convert a broad range of renewable heterotrophic resources into diverse valuable compounds. High cell density fermentations of the non-pathogenic R. eutropha can be easily performed. Furthermore, this bacterium is accessible to engineering of its metabolism by genetic approaches having available a large repertoire of genetic tools. Since the complete genome sequence of R. eutropha H16 has become available, a variety of transcriptome, proteome and metabolome studies provided valuable data elucidating its complex metabolism and allowing a systematic biology approach. However, high production costs for bacterial large-scale production of biomass and biotechnologically valuable products are still an economic challenge. The application of inexpensive raw materials could significantly reduce the expenses. Therefore, the conversion of diverse substrates to polyhydroxyalkanoates by R. eutropha was steadily improved by optimization of cultivation conditions, mutagenesis and metabolic engineering. Industrial by-products and residual compounds like glycerol, and substrates containing high carbon content per weight like palm, soybean, corn oils as well as raw sugar-rich materials like molasses, starch and lignocellulose, are the most promising renewable substrates and were intensively studied.

  15. Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments.

    PubMed

    Mijnendonckx, K; Provoost, A; Ott, C M; Venkateswaran, K; Mahillon, J; Leys, N; Van Houdt, R

    2013-02-01

    Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C(254nm) radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5-4 μM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 μM AgNO(3) in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.

  16. Production of Poly (3-Hydroxybutyric Acid) by Ralstonia eutropha in a Biocalorimeter and its Thermokinetic Studies.

    PubMed

    Anusha, Subramanian Mohanakrishnan; Leelaram, Santharam; Surianarayanan, Mahadevan

    2016-07-01

    Bioplastic production from microbial sources is an emerging area which provides opportunities even to convert the wastes into bioplastics. Poly (3-hydroxybutyric acid), commonly called as PHB, is a bioplastic, which is stored as intracellular cytoplasmic inclusions in microorganisms. The objectives of this study are to calorimetrically monitor the PHB production and evaluate the thermokinetic data in a bioreaction calorimeter (BioRC1e). Thus, a well-known PHB-producing bacteria Ralstonia eutropha was selected for batch process in a bioreaction calorimeter. The metabolic heat generated was found to be correlated with the biomass, substrate consumption, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and PHB production. The OUR pattern explained the oxidative metabolism of the strain R. eutropha. The heat yields due to biomass and glucose consumption during PHB production were found to be 12.56 and 13.56 kJ/g, respectively. The oxycalorific value obtained for the PHB production was 443.80 kJ/mol of O2. The concentration of PHB obtained in BioRC1e was 4.33 g/L with a production rate of 0.09 g/L/h. The chemical structure of the extracted PHB by R. eutropha was confirmed using fourier transform infrared spectroscopy (FT-IR) and (1)H and (13)C nuclear magnetic resonance (NMR) analysis.

  17. Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

    SciTech Connect

    Lu, JN; Brigham, CJ; Gai, CS; Sinskey, AJ

    2012-08-04

    Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.

  18. Metabolic engineering of Ralstonia eutropha for the production of polyhydroxyalkanoates from sucrose.

    PubMed

    Park, Si Jae; Jang, Young-Ah; Noh, Won; Oh, Young Hoon; Lee, Hyuk; David, Yokimiko; Baylon, Mary Grace; Shin, Jihoon; Yang, Jung Eun; Choi, So Young; Lee, Seung Hwan; Lee, Sang Yup

    2015-03-01

    A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes β-fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that β-fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20 g/L of sucrose, a high P(3HB) content of 73.2 wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5 mol% LA) to a polymer content of 19.5 wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5 mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52 × 10(5) (Mn ) and 2.19 × 10(4) (Mn ), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources.

  19. Comparison of Candiatus Liberibacter solanacearum genomic sequences isolated from carrot psyllids in Scandinavia and potato psyllids from Texas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fastidious prokaryote Candidatus Liberibacter solanacearum (Lso), transmitted by the tomato potato psyllid (Bactericera cockerelli), is associated with the Zebra Chip disease of potato. Plants infected with Liberibacter may experience significant yield losses and these plants also serve as poten...

  20. Natural products from environmental DNA hosted in Ralstonia metallidurans.

    PubMed

    Craig, Jeffrey W; Chang, Fang-Yuan; Brady, Sean F

    2009-01-16

    Metagenomic studies designed to access new small molecules from the heterologous expression of environmental DNA have focused on the use of two model systems, Escherichia coli and Streptomyces spp., as heterologous hosts. Accessing the biosynthetic potential of DNA extracted from the bacteria present in environmental samples will require the development of a more diverse collection of model bacterial hosts that can be used for screening environmental DNA libraries. In this study the bacterium Ralstonia metallidurans was explored as a heterologous host. Here we report the isolation and characterization of both novel and known metabolites from pigmented and antibacterially active clones found in R. metallidurans based environmental DNA libraries. The clones found in this study do not confer the production of clone-specific metabolites to E. coli, validating R. metallidurans as an orthogonal expression host that can be used to expand the number of metabolites found in future metagenomic discovery efforts.

  1. 78 FR 25939 - Submission for OMB Review; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-03

    ... Program for Imported Articles to Prevent Introduction of Potato Brown Rot. OMB Control Number: 0579-0221... bacterium Ralstonia solanacearum race 3 biovar 2 is known to occur. This bacterial strain causes potato brown rot, which causes potatoes to rot through, making them inedible and seriously affecting...

  2. A case study of a bacterial pathogen in irrigation water

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter presents a case study of how exotic strains of Ralstonia solanacearum were disseminated throughout Europe and Florida via waterways used for irrigation. Several studies have demonstrated that aquatic weeds that commonly grow in rivers and ponds are able to harbor the pathogen and allow ...

  3. Regulation of extracellular polygalacturonase production in Pseudomonas solanacearum. Progress report, [May 1, 1992--April 30, 1994

    SciTech Connect

    Allen, C.

    1994-06-01

    Pseudomonas solanacearum is an economically important plant pathogen that causes bacterial wilt disease of diverse crops. The bacterium produces at least three isozymes of polygalacturonase, which degrade plant cell walls and contribute substantially to bacterial wilt disease development. The central objective of this research project is to determine how expression of these enzymes is regulated. To this end, we isolated a positive trans-acting regulator of polygalacturonase production (pehR). We have focused on further characterization of the pehR mutant pheonotype, and studies of pehR expression. Preliminary results suggest pehR also regulates bacterial motility. An investigation of two unusual tyrosine phosphoproteins in P. solanacearum is also described.

  4. Synergistic interaction in dual-species biofilms formation by Escherichia coli O157:H7 and Ralstonia spp

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Ralstonia spp., a heterotrophic bacterium that are isolated from produce processing environments as part of the native microflora, have strong potentials for formaing biofilms on various surfaces. When co-cultured, Escherichia coli O157:H7 (EcO157) and Ralstonia spp. displayed a synerg...

  5. Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

    PubMed Central

    Ushimaru, Kazunori; Mizuno, Shoji

    2015-01-01

    Recombinant Ralstonia eutropha strain PHB−4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB−4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB−4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone. PMID:25645560

  6. "Candidatus Liberibacter solanacearum" Associated With the Psyllid, Bactericera maculipennis (Hemiptera: Triozidae).

    PubMed

    Borges, Karina M; Cooper, W Rodney; Garczynski, Stephen F; Thinakaran, Jenita; Jensen, Andy S; Horton, David R; Munyaneza, Joseph E; Cueva, Isabel; Barcenas, Nina M

    2017-01-20

    The psyllid Bactericera maculipennis (Crawford) (Hemiptera: Triozidae) often cohabits field bindweed (Convolvulus arvensis, Solanales: Convolvulaceae) and other plants with the congeneric psyllid, Bactericera cockerelli (Šulc), in the Pacific Northwestern United States. Bactericera cockerelli is a vector of "Candidatus Liberibacter solanacearum," the pathogen associated with zebra chip disease of potato (Solanales: Solanaceae). Because B. maculipennis and B. cockerelli both naturally occur on certain plants, we surveyed B. maculipennis adults collected from Washington and Idaho for presence of "Ca. L. solanacearum" to determine whether this psyllid also harbors this pathogen. Liberibacter was present in 30% of field-collected B. maculipennis and in 100% of colony-reared psyllids. Sequences of 16S rDNA and microsatellite markers revealed that "Ca. L. solanacearum" from B. maculipennis was closely related to Liberibacter haplotype B from B. cockerelli Results of laboratory assays demonstrated that Liberibacter can be transmitted between B. cockerelli and B. maculipennis on plants within the Convolvulaceae. Potato plants challenged with Liberibacter-infected B. maculipennis did not become infected, apparently because potato is not a suitable host for the psyllid. We therefore conclude that B. maculipennis is not a direct threat to potato production, despite its association with Liberibacter. We are the first to report that "Ca. L. solanacearum" is associated with a psyllid other than B. cockerelli in North America. Results of our study demonstrate the importance of understanding the complete ecology of psyllids-including interactions with other psyllids on non-crop hosts-in predicting what crops or regions are potentially susceptible to the spread of Liberibacter.

  7. An Outbreak of Ralstonia pickettii Bloodstream Infection Associated with an Intrinsically Contaminated Normal Saline Solution.

    PubMed

    Chen, Yin-Yin; Huang, Wan-Tsuei; Chen, Chia-Ping; Sun, Shu-Mei; Kuo, Fu-Mei; Chan, Yu-Jiun; Kuo, Shu-Chen; Wang, Fu-Der

    2017-04-01

    OBJECTIVE Ralstonia pickettii has caused contamination of pharmaceutical solutions in many countries, resulting in healthcare infections or outbreak events. We determined the source of the outbreak of R. pickettii bloodstream infection (BSI). METHODS This study was conducted in a 3,000-bed tertiary referral medical center in Taiwan with >8,500 admissions during May 2015. Patients had been treated in the injection room or chemotherapy room at outpatient departments, emergency department, or hospital wards. All patients who were culture positive for R. pickettii from May 3 to June 11, 2015, were eligible for the study. The aim of the survey was to conduct clinical epidemiological and microbiological investigations to identify possible sources of infection. RESULTS We collected 57 R. pickettii-positive specimens from 30 case patients. We performed 24 blood cultures; 14 of these revealed >2 specimens and 6 used fluid withdrawn from Port-a-Cath implantable venous access devices. All patients received an injection of 20 mL 0.9% normal saline via catheter flushing. In addition, 2 unopened ampules of normal saline solution (20 mL) were confirmed positive for R. pickettii. The Taiwan Centers for Disease Control and Prevention performed sampling and testing of the same manufactured batch and identified the same strain of R. pickettii. Pulsed-field gel electrophoresis tests revealed that all clinical isolates had similarity of >90%, validating the outbreak of the same clone of R. pickettii. CONCLUSIONS R. pickettii can grow in saline solutions and cause bloodstream infections. Hospital monitoring mechanisms are extremely important measures in identifying and ending such outbreaks. Infect Control Hosp Epidemiol 2017;38:444-448.

  8. Highly sensitive and selective gold(I) recognition by a metalloregulator in Ralstonia metallidurans.

    PubMed

    Jian, Xing; Wasinger, Erik C; Lockard, Jenny V; Chen, Lin X; He, Chuan

    2009-08-12

    A MerR family metalloregulatory protein CupR selectively responds to gold stress in Ralstonia metallidurans. A distorted trigonal geometry appears to be used by CupR to achieve the highly sensitive (K(d) approximately 10(-35) M) and selective recognition of gold(I).

  9. A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia eutropha is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and the molecular engineering of this organism. We have developed a versatile...

  10. Association of "Candidatus Liberibacter solanacearum" with the psyllid, Trioza apicalis (Hemiptera: Triozidae) in Europe.

    PubMed

    Munyaneza, Joseph E; Fisher, Tonja W; Sengoda, Venkatesan G; Garczynski, Stephen F; Nissinen, Anne; Lemmetty, Anne

    2010-08-01

    The psyllid Trioza apicalis Förster (Hemiptera: Triozidae) is a serious pest of carrots, Daucus carota L., in Europe. Carrots exhibiting symptoms of psyllid damage were observed in commercial fields in southern Finland in 2008. Symptoms in affected plants included leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots. Mechanisms by which T. apicalis induces symptoms in plants are not understood, and no plant pathogens have yet been associated with this insect. Given recent association of liberibacter with several crops affected by psyllids, an investigation on whether this bacterium is associated with T. apicalis was conducted. Polymerase chain reaction (PCR) primer pairs OA2/OI2c and LsoF/OI2c, specific for 16S rRNA gene from "Candidatus Liberibacter solanacearum," generated amplicons of 1,168 bp and 1,173 bp, respectively, from DNA extracted from field-collected psyllids (61 and 36.6%, respectively), laboratory-reared psyllids (70 and 33.3%, respectively), field-collected petioles from symptomatic carrots (80 and 55%, respectively), and laboratory-grown carrots (100% for both primer pairs). In contrast, no PCR products were detected in DNA extracted from insect-free plants. The DNA sequences of amplicons of the genes encoding liberibacter 16S rRNA from psyllids and carrots were identical. DNA of the 16S rRNA gene sequences determined from carrots and psyllids were 99.9% identical to analogous sequences of "Ca. L. solanacearum" amplified from several solanaceous crops and the psyllid Bactericera cockerelli (Sulc), a vector of this bacterium. This is the first report of a plant pathogen associated with T. apicalis and the second known psyllid species associated with "Ca. L. solanacearum".

  11. Intraspecific Variability within Globodera tabacum solanacearum Using Random Amplified Polymorphic DNA

    PubMed Central

    Syracuse, A. J.; Johnson, C. S.; Eisenback, J. D.; Nessler, C. L.; Smith, E. P.

    2004-01-01

    Random amplified polymorphic DNA (RAPDs) were used to investigate the intraspecific variability among 19 geographic isolates of Globodera tabacum solanacearum from eight counties in Virginia and one county in North Carolina. Globodera tabacum tabacum, G. t. virginiae, and the Mexican cyst nematode (MCN) were included as outgroups. Six primers were used and 119 amplification products were observed. Each primer yielded reproducible differences in fragment patterns that differentiated the isolates and species. Hierarchical cluster analysis was performed to illustrate the relatedness among isolates and species. The average Jaccard's similarity index among isolates of G. t. solanacearum was 74%, possibly representing greater variation than that reported in the literature across different pathotypes of the potato cyst nematode, Globodera pallida, in studies where RAPD were also employed. The RAPD markers described here may be useful for the development of specific primers or probes that could improve the identification of TCN populations. Such improvements in the characterization of TCN genotypes would facilitate the effective deployment of existing and future resistant cultivars to control these economically important pests. PMID:19262823

  12. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

    SciTech Connect

    Lu, JN; Brigham, CJ; Plassmeier, JK; Sinskey, AJ

    2014-08-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K-M = 10.5 mu M) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2

  13. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16.

    PubMed

    Pötter, Markus; Madkour, Mohamed H; Mayer, Frank; Steinbüchel, Alexander

    2002-08-01

    Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R. eutropha. The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of R. eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules. PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the -10 region and a region immediately upstream of the -35 region of the sigma(70) promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the phaR translational start codon, where the sigma(54)-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a phaROmegaKm mutant of R. eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of phaP expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules

  14. Identification of a novel Gsp-related pathway required for secretion of the manganese-oxidizing factor of Pseudomonas putida strain GB-1.

    PubMed

    De Vrind, Johannes; De Groot, Arjan; Brouwers, Geert Jan; Tommassen, Jan; De Vrind-De Jong, Elisabeth

    2003-02-01

    The manganese-oxidizing factor of Pseudomonas putida strain GB-1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two-gene operon that includes an xcpS homologue (designated xcmS). No other xcp-like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB-1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin-related protein. This xcmX is clustered with two other xcpT-related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well-described 'regular'xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp-related system for the transport of manganese-oxidizing enzymes to the cell surface.

  15. Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB storage in recombinant Escherichia coli.

    PubMed

    Eggers, Jessica; Steinbüchel, Alexander

    2014-12-01

    The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay

  16. DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum.

    PubMed Central

    Huang, J H; Schell, M A

    1990-01-01

    The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum. The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined. A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato. Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence. Localization of various pglA-phoA fusion proteins in Escherichia coli and P. solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria. Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P. solanacearum cell. Our results suggest that PGA excretion occurs in two steps. The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E. coli. Images PMID:2193922

  17. Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage

    PubMed Central

    Chen, Janice S.; Colón, Brendan; Dusel, Brendon; Ziesack, Marika; Torella, Joseph P.

    2015-01-01

    Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha. PMID:26664804

  18. Biological and chemical induction of resistance to the Globodera tabacum solanacearum in oriental and flue-cured tobacco (Nicotiana tabacum L.).

    PubMed

    Parkunan, Venkatesan; Johnson, Charles S; Eisenback, Jon D

    2009-09-01

    The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field.

  19. Biological and Chemical Induction of Resistance to the Globodera tabacum solanacearum in Oriental and Flue-Cured Tobacco (Nicotiana tabacum L.)

    PubMed Central

    Johnson, Charles S.; Eisenback, Jon D.

    2009-01-01

    The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field. PMID:22736815

  20. Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp.CS1 and Ralstonia sp. CS2 isolated from agricultural soils.

    PubMed

    Smejkal, C W; Vallaeys, T; Seymour, F A; Burton, S K; Lappin-Scott, H M

    2001-04-01

    The herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad-leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade (R/S)-mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4-D (2,4-dichlorophenoxyacetic acid), MCPA [4-chloro-2-methyl phenoxy acetic acid] and (R/S)-2,4-DP [2-(2,4-dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene-specific probes derived from the 2,4-D-degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd-like genes are present in mecoprop-degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA, tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA, tfdB and tfdC genes of V. paradoxus. It is therefore likely that tfd-like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.

  1. Ralstonia pickettii and Burkholderia cepacia complex bloodstream infections related to infusion of contaminated water for injection.

    PubMed

    Moreira, B M; Leobons, M B G P; Pellegrino, F L P C; Santos, M; Teixeira, L M; de Andrade Marques, E; Sampaio, J L M; Pessoa-Silva, C L

    2005-05-01

    Ralstonia pickettii and Burkholderia cepacia complex isolates are causes of healthcare-associated infection related to contamination of intravenously administered products. Based on microbiological and epidemiological data and molecular typing by pulsed-field gel electrophoresis, we report the occurrence of two outbreaks of R. pickettii and B. cepacia complex bloodstream infections. The first outbreak occurred from August 1995 to September 1996, and the second outbreak occurred from 28 March to 8 April 1998, affecting adults and neonates, respectively. Infusion of contaminated water for injection was the source of infection.

  2. Association of 'Candidatus Liberibacter solanacearum' with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection.

    PubMed

    Teresani, Gabriela R; Bertolini, Edson; Alfaro-Fernández, Ana; Martínez, Carmen; Tanaka, Francisco André Ossamu; Kitajima, Elliot W; Roselló, Montserrat; Sanjuán, Susana; Ferrándiz, Juan Carlos; López, María M; Cambra, Mariano; Font, María Isabel

    2014-08-01

    A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

  3. Effects of Host Resistance and Temperature on Development of Globodera tabacum solanacearum

    PubMed Central

    Wang, J.; Johnson, C. S.; Eisenback, J. D.

    2001-01-01

    Penetration and development of juveniles of tobacco cyst nematode (Globodera tabacum solanacearum) on a resistant (NC567) and a susceptible (K326) cultivar of flue-cured tobacco (Nicotiana tabacum L.) were determined in root zone chamber experiments. More vermiform juveniles developed into a swollen shape at 22, 27, and 31 °C than at 17 °C. Development of flask-shaped nematodes appeared to be similar across tested temperatures (17, 22, 27, and 31 °C). General patterns of penetration and development of juveniles in both resistant and susceptible cultivars were similar under all temperatures tested. More vermiform, swollen, and flask-shaped nematodes were found in roots of K326 than in those of NC567. Development from swollen to flaskshaped nematodes appeared to be similar between the two cultivars, although more vermiform juveniles developed into swollen nematodes on K326 than on NC567. Differences in resistance between the two cultivars remained stable across tested temperatures. PMID:19266009

  4. Influence of the pathogen Candidatus Liberibacter solanacearum on tomato host plant volatiles and psyllid vector settlement.

    PubMed

    Mas, Flore; Vereijssen, Jessica; Suckling, David M

    2014-12-01

    Candidatus Liberibacter solanacearum (CLso) is an unculturable bacterium vectored by the tomato potato psyllid (TPP) Bactericera cockerelli and has been associated with Zebra chip disease in potato and with other economically relevant symptoms observed in solanaceous crops. By altering their host and vector's biological system, pathogens are able to induce changes that benefit them by increasing their transmission rate. Understanding these changes can enable better targeting of mechanisms to control pathogen outbreaks. Here, we explored how the CLso infectious status affects the volatile organic compounds (VOCs) of the tomato plant, and whether the CLso infectious status of TPP influences host plant settlement. These chemical and behavioral changes can ultimately affect the rate of encounter between the host and the vector. Results from headspace volatile collection of tomato plants showed that CLso infected tomato plants emitted a qualitatively and quantitatively different blend of VOCs compared to sham-infected plants. By a factorial experiment, we showed that CLso negative (CLso-) TPP preferred to settle 70 % more often on infected tomato plants, while CLso positive (CLso+) TPP were found 68 % more often on sham-infected tomato plants. These results provide new evidence in favor of both host and vector manipulation by CLso.

  5. Colonization and Intrusive Invasion of Potato Psyllid by 'Candidatus Liberibacter solanacearum'.

    PubMed

    Cicero, Joseph M; Fisher, Tonja W; Qureshi, Jawwad A; Stansly, Philip A; Brown, Judith K

    2017-01-01

    Previous studies have shown that the fastidious bacterial plant pathogen 'Candidatus Liberibacter solanacearum' (CLso) is transmitted circulatively and propagatively by the potato psyllid (PoP) Bactericera cockerelli. In this study, the temporal and spatial interrelationships between CLso PoP were investigated by scanning electron microscopy of the digestive system of PoP immature and adult instars and salivary glands of adults post CLso ingestion. CLso biofilms were not detectable on the outer midgut surface of the first and second instars; however, for third to fifth instars and teneral and mature adults, biofilms were observed in increasing numbers in each successive developmental stage. In adult PoP midguts, CLso cells were observed between the basal lamina and basal epithelial cell membranes; in basal laminar perforations, on the outer basal laminar surface, and in the ventricular lumen, epithelial cytosol, and filter chamber periventricular space. CLso were also abundantly visible in the salivary gland pericellular spaces and in the epidermal cell cytosol of the head. Collectively, these results point to an intrusive, systemic invasion of PoP by CLso that employs an endo/exocytosis-like mechanism, in the context of a propagative, circulative mode of transmission.

  6. Implications of various phosphoenolpyruvate-carbohydrate phosphotransferase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16

    PubMed Central

    2011-01-01

    The enhanced global biodiesel production is also yielding increased quantities of glycerol as main coproduct. An effective application of glycerol, for example, as low-cost substrate for microbial growth in industrial fermentation processes to specific products will reduce the production costs for biodiesel. Our study focuses on the utilization of glycerol as a cheap carbon source during cultivation of the thermoplastic producing bacterium Ralstonia eutropha H16, and on the investigation of carbohydrate transport proteins involved herein. Seven open reading frames were identified in the genome of strain H16 to encode for putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS). Although the core components of PEP-PTS, enzyme I (ptsI) and histidine phosphocarrier protein (ptsH), are available in strain H16, a complete PTS-mediated carbohydrate transport is lacking. Growth experiments employing several PEP-PTS mutants indicate that the putative ptsMHI operon, comprising ptsM (a fructose-specific EIIA component of PTS), ptsH, and ptsI, is responsible for limited cell growth and reduced PHB accumulation (53%, w/w, less PHB than the wild type) of this strain in media containing glycerol as a sole carbon source. Otherwise, the deletion of gene H16_A0384 (ptsN, nitrogen regulatory EIIA component of PTS) seemed to largely compensate the effect of the deleted ptsMHI operon (49%, w/w, PHB). The involvement of the PTS homologous proteins on the utilization of the non-PTS sugar alcohol glycerol and its effect on cell growth as well as PHB and carbon metabolism of R. eutropha will be discussed. PMID:21906371

  7. Seasonal population dynamics of the potato psyllid (Hemiptera: Triozidae) and its associated pathogen "Candidatus Liberibacter solanacearum" in potatoes in the southern great plains of North America.

    PubMed

    Goolsby, J A; Adamczyk, J J; Crosslin, J M; Troxclair, N N; Anciso, J R; Bester, G G; Bradshaw, J D; Bynum, E D; Carpio, L A; Henne, D C; Joshi, A; Munyaneza, J E; Porter, P; Sloderbeck, P E; Supak, J R; Rush, C M; Willett, F J; Zechmann, B J; Zens, B A

    2012-08-01

    The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), and its associated pathogen "Candidatus Liberibacter solanacearum" (Ca. L. solanacearum), the putative causal agent of zebra chip (ZC) disease in potatoes (Solanum tuberosum L.), were sampled in commercial potato fields and untreated control plots for 3 yr in multiple locations in Texas, Kansas, Nebraska, and Colorado. Populations of the potato psyllid varied across years and across potato growing regions. However, the percentage of potato psyllids infected with Ca. L. solanacearum although variable across years, was consistently highest in the Lower Rio Grande Valley of Texas (LRGV), the reported overwintering location for this pest. The numbers of Ca. L. solanacearum-infected psyllids collected on field traps and large nymphs counted on leaf samples were both positively correlated with the final percentage of ZC in tubers. In the LRGV, where vector and disease pressure is the highest, population levels of immature life stages of the psyllid and percentage of ZC differed greatly between commercial and untreated fields. These results show that the pest management program that was used can be effective at controlling development of the psyllid and ultimately reducing the incidence of ZC.

  8. A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

    PubMed Central

    Srinivasan, Sriram; Barnard, Gavin C.; Gerngross, Tillman U.

    2002-01-01

    We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli. PMID:12450812

  9. Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16.

    PubMed

    Pohlmann, Anne; Fricke, Wolfgang Florian; Reinecke, Frank; Kusian, Bernhard; Liesegang, Heiko; Cramm, Rainer; Eitinger, Thomas; Ewering, Christian; Pötter, Markus; Schwartz, Edward; Strittmatter, Axel; Voss, Ingo; Gottschalk, Gerhard; Steinbüchel, Alexander; Friedrich, Bärbel; Bowien, Botho

    2006-10-01

    The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.

  10. Large scale extraction of poly(3-hydroxybutyrate) from Ralstonia eutropha H16 using sodium hypochlorite.

    PubMed

    Heinrich, Daniel; Madkour, Mohamed H; Al-Ghamdi, Mansour A; Shabbaj, Ibraheem I; Steinbüchel, Alexander

    2012-11-19

    Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes.

  11. Large scale extraction of poly(3-hydroxybutyrate) from Ralstonia eutropha H16 using sodium hypochlorite

    PubMed Central

    2012-01-01

    Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes. PMID:23164136

  12. Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production

    PubMed Central

    2013-01-01

    Background The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5’ mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well. PMID:24219429

  13. Biological control of bacterial wilt in Arabidopsis thaliana involves abscissic acid signalling.

    PubMed

    Feng, Dong Xin; Tasset, Céline; Hanemian, Mathieu; Barlet, Xavier; Hu, Jian; Trémousaygue, Dominique; Deslandes, Laurent; Marco, Yves

    2012-06-01

    Means to control bacterial wilt caused by the phytopathogenic root bacteria Ralstonia solanacearum are limited. Mutants in a large cluster of genes (hrp) involved in the pathogenicity of R. solanacearum were successfully used in a previous study as endophytic biocontrol agents in challenge inoculation experiments on tomato. However, the molecular mechanisms controlling this resistance remained unknown. We developed a protection assay using Arabidopsis thaliana as a model plant and analyzed the events underlying the biological control by genetic, transcriptomic and molecular approaches. High protection rates associated with a significant decrease in the multiplication of R. solanacearum were observed in plants pre-inoculated with a ΔhrpB mutant strain. Neither salicylic acid, nor jasmonic acid/ethylene played a role in the establishment of this resistance. Microarray analysis showed that 26% of the up-regulated genes in protected plants are involved in the biosynthesis and signalling of abscissic acid (ABA). In addition 21% of these genes are constitutively expressed in the irregular xylem cellulose synthase mutants (irx), which present a high level of resistance to R. solanacearum. We propose that inoculation with the ΔhrpB mutant strain generates a hostile environment for subsequent plant colonization by a virulent strain of R. solanacearum.

  14. Transcriptome analyses of Bactericera cockerelli adults in response to "Candidatus Liberibacter solanacearum" infection.

    PubMed

    Nachappa, Punya; Levy, Julien; Tamborindeguy, Cecilia

    2012-10-01

    The potato/tomato psyllid, Bactericera cockerelli (Šulc) is an economically important crop pest that not only causes damage through its feeding but also transmits the bacterium, "Candidatus Liberibacter solanacearum" (CLs), which causes zebra chip disease in potato. There is some information about the phenotypic effects of phytopathogenic bacteria on their insect vectors; however, there are no published reports of the molecular mechanisms underlying phytopathogenic bacteria-insect vector interaction. In order to investigate the effects of CLs infection on B. cockerelli, transcriptomic analyses of CLs-infected and uninfected adult psyllids that were reared on potato were performed. De novo assembly of cDNA sequences generated 136,518 and 109,983 contigs for infected and uninfected insect libraries with an average contig length of 514 bp. BlastX analysis against the NCBI-nr database revealed that 33.33 % had significant matches. Gene ontology data illustrated that the majority of the expressed psyllid genes are involved in metabolic process, biological regulation, binding and catalytic activity. The psyllid transcriptome had an abundance of genes such as vitellogenin, heat shock protein, ejaculatory bulb-specific protein, ferritin, and cytochrome oxidase. Notably absent in the psyllid transcriptome were innate immunity genes induced in response to Gram-negative bacteria (IMD pathway). Several functionally diverse contigs related to symbiotic bacteria including the primary endosymbiont Carsonella ruddii, Wolbachia, and CLs in the psyllid transcriptome were identified. A total of 247 contigs showed differential expression in response to CLs infection including immune and stress-related genes and vitellogenins. Expression analyses of selected psyllid genes were performed on psyllids that were exclusively reared on potato (host of the insects used for RNAseq) and psyllids exclusively reared on tomato (alternative host of psyllids). These genes showed similar expression

  15. Construction of a bioluminescent reporter strain to detect polychlorinated biphenyls

    SciTech Connect

    Layton, A.C.; Muccini, M.; Ghosh, M.M.; Sayler, G.S.

    1998-12-01

    A bioluminescent reporter strain, Ralstonia eutropha ENV307 (pUTK60), was constructed for the detection of polychlorinated biphenyls by inserting the biphenyl promoter upstream of the bioluminescence genes. In the presence of a nonionic surfactant, which enhances the solubility of chlorinated biphenyls, bioluminescence was induced three- to fourfold over background by biphenyl, monochlorinated biphenyls, and Aroclor 1242. The minimum detection limits for these compounds ranged from 0.15 mg/liter for 4-chlorobiphenyl to 1.5 mg/liter for Aroclor 1242.

  16. Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from Carbon Dioxide, Hydrogen, and Oxygen Project Final Report

    SciTech Connect

    Sinskey, Anthony J.; Worden, Robert Mark; Brigham, Christopher; Lu, Jingnan; Quimby, John Westlake; Gai, Claudia; Speth, Daan; Elliott, Sean; Fei, John Qiang; Bernardi, Amanda; Li, Sophia; Grunwald, Stephan; Grousseau, Estelle; Maiti, Soumen; Liu, Chole

    2013-12-16

    This research project is a collaboration between the Sinskey laboratory at MIT and the Worden laboratory at Michigan State University. The goal of the project is to produce Isobutanol (IBT), a branched-chain alcohol that can serve as a drop-in transportation fuel, through the engineered microbial biosynthesis of Carbon Dioxide, Hydrogen, and Oxygen using a novel bioreactor. This final technical report presents the findings of both the biological engineering work at MIT that extended the native branched-chain amino acid pathway of the wild type Ralstonia eutropha H16 to perform this biosynthesis, as well as the unique design, modeling, and construction of a bioreactor for incompatible gasses at Michigan State that enabled the operational testing of the complete system. This 105 page technical report summarizing the three years of research includes 72 figures and 11 tables of findings. Ralstonia eutropha (also known as Cupriavidus necator) is a Gram-negative, facultatively chemolithoautotrophic bacteria. It has been the principle organism used for the study of polyhydroxybutyrate (PHB) polymer biosynthesis. The wild-type Ralstonia eutropha H16 produces PHB as an intracellular carbon storage material while under nutrient stress in the presence of excess carbon. Under this stress, it can accumulate approximately 80 % of its cell dry weight (CDW) as this intracellular polymer. With the restoration of the required nutrients, the cells are then able to catabolize this polymer. If extracted from the cell, this PHB polymer can be processed into biodegradable and biocompatible plastics, however for this research, it is the efficient metabolic pathway channeling the captured carbon that is of interest. R. eutropha is further unique in that it contains two carbon-fixation Calvin–Benson–Bassham cycle operons, two oxygen-tolerant hydrogenases, and several formate dehydrogenases. It has also been much studied for its ability in the presence of oxygen, to fix carbon dioxide

  17. A gene cluster required for coordinated biosynthesis of lipopolysaccharide and extracellular polysaccharide also affects virulence of Pseudomonas solanacearum.

    PubMed Central

    Kao, C C; Sequeira, L

    1991-01-01

    Bacterial cell surface components can be important determinants of virulence. At least three gene clusters important for extracellular polysaccharide (EPS) biosynthesis have been previously identified in the plant pathogen Pseudomonas solanacearum. We have found that one of these gene clusters, named ops, is also required for lipopolysaccharide (LPS) biosynthesis. Mutations in any complementation unit of this cluster decreased EPS production, prevented the binding of an LPS-specific phage, and altered the mobility of purified LPS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, restoration of LPS biosynthesis alone was not sufficient to restore virulence to the wild-type level, suggesting that EPS is important for pathogenesis. Images FIG. 2 FIG. 3 PMID:1744040

  18. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    PubMed Central

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  19. Dynamic mathematical models for biodegradation of formaldehyde by Ralstonia eutropha in a batch bioreactor.

    PubMed

    Habibi, Alireza; Vahabzadeh, Farzaneh; Zaiat, Marcelo

    2013-11-15

    Degradation of formaldehyde by Ralstonia eutropha was studied in a batch bioreactor operated in recycling mode (30 °C, initial pH of 6.5, aeration rate 0.5 vvm, and a recycling flow rate of 6 mL min(-1)). Growth kinetics equations were described using four substrate inhibition models, and the initial formaldehyde concentration ranged from 54.5 to 993.0 mg L(-1). In each case, model parameters were estimated interactively using nonlinear regression analysis and on the basis of the goodness of fit, the fitness of the model to the experimental data was obtained (i.e., the coefficient of determination and the percent of standard deviation). The estimated parameters according to the Luong equation were μmax = 0.101 h(-1), KS = 54.1 mg L(-1), Sm = 1329 mg L(-1), and n = 2.07. According to the maintenance energies explained by Pirt, cell maintenance was quantified with q = Aμ + B; where A and B are the associated and non-associated growth parts of substrate consumption, respectively. The importance of these terms was verified using the developed models, which would efficiently describe the dynamic nature of growth and formaldehyde biodegradation.

  20. Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

    PubMed Central

    Müller, Jana; MacEachran, Daniel; Burd, Helcio; Sathitsuksanoh, Noppadon; Bi, Changhao; Yeh, Yi-Chun; Lee, Taek Soon; Hillson, Nathan J.; Chhabra, Swapnil R.; Singer, Steven W.

    2013-01-01

    Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones. PMID:23686271

  1. Investigation of the NADH/NAD(+) ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox.

    PubMed

    Tejwani, Vijay; Schmitt, Franz-Josef; Wilkening, Svea; Zebger, Ingo; Horch, Marius; Lenz, Oliver; Friedrich, Thomas

    2017-01-01

    Ralstonia eutropha is a hydrogen-oxidizing ("Knallgas") bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H2-driven production of biodegradable polymers and hydrocarbons. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H2 oxidation with the reduction of NAD(+) to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD(+) pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD(+)] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD(+)] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD(+)] ratios represents a novel and sensitive tool to determine the redox state of cells.

  2. Crystal structure of Ralstonia eutropha polyhydroxyalkanoate synthase C-terminal domain and reaction mechanisms.

    PubMed

    Kim, Jieun; Kim, Yeo-Jin; Choi, So Young; Lee, Sang Yup; Kim, Kyung-Jin

    2017-01-01

    Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by numerous microorganisms as energy and reducing power storage materials, and have attracted much attention as substitutes for petroleum-based plastics. Here, we report the first crystal structure of Ralstonia eutropha PHA synthase at 1.8 Å resolution and structure-based mechanisms for PHA polymerization. RePhaC1 contains two distinct domains, the N-terminal (RePhaC1ND ) and C-terminal domains (RePhaC1CD ), and exists as a dimer. RePhaC1CD catalyzes polymerization via non-processive ping-pong mechanism using a Cys-His-Asp catalytic triad. Molecular docking simulation of 3-hydroxybutyryl-CoA to the active site of RePhaC1CD reveals residues involved in the formation of 3-hydroxybutyryl-CoA binding pocket and substrate binding tunnel. Comparative analysis with other polymerases elucidates how different classes of PHA synthases show different substrate specificities. Furthermore, we attempted structure-based protein engineering and developed a RePhaC1 mutant with enhanced PHA synthase activity.

  3. A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha.

    PubMed

    Solaiman, Daniel K Y; Swingle, Bryan M; Ashby, Richard D

    2010-08-01

    Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB(-)4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4x10(3) Km-resistance colonies/mug DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor alpha-galactosidase in R. eutropha.

  4. Phenol degradation by Ralstonia eutropha: Colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations

    SciTech Connect

    Leonard, D; Destruhaut, C; Lindley, N D; Youssef, C B; Queinnec, I

    1999-11-20

    Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a K[sub s] of 2 mg/L, a K[sub i] of 350 mg/L and a [micro][sub max] of 0.41 h[sup [minus]1]. Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2phms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled. This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h[sup [minus]1], close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m[sup 3]/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.

  5. Effects of Homologous Phosphoenolpyruvate-Carbohydrate Phosphotransferase System Proteins on Carbohydrate Uptake and Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha H16▿†

    PubMed Central

    Kaddor, Chlud; Steinbüchel, Alexander

    2011-01-01

    Seven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome of Ralstonia eutropha H16 by in silico analysis. Except the N-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACB and H16 ΔnagFEC mutants exhibited no growth when cultivated on fructose and N-acetylglucosamine, respectively. Furthermore, a transposon mutant with a ptsM-ptsH insertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mob insertion. ptsM, ptsH, and ptsI single, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384, frcACB, or nagFEC genes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of the ptsMHI deletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins in R. eutropha H16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism. PMID:21478317

  6. Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

    PubMed Central

    Schäfer, Caspar; Friedrich, Bärbel

    2013-01-01

    Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2. PMID:23793632

  7. Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

    PubMed Central

    Wenning, Leonie; Stöveken, Nadine; Wübbeler, Jan Hendrik

    2015-01-01

    Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs. PMID:26590284

  8. Biosynthesis and thermal properties of PHBV produced from levulinic acid by Ralstonia eutropha.

    PubMed

    Wang, Yuanpeng; Chen, Ronghui; Cai, JiYuan; Liu, Zhenggui; Zheng, Yanmei; Wang, Haitao; Li, Qingbiao; He, Ning

    2013-01-01

    Levulinic acid (LA) can be cost-effectively produced from a vast array of renewable carbohydrate-containing biomaterials. LA could facilitate the commercialization of the polymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and PHBV-based products as carbon substrates. Therefore, this paper focused on the production of PHBV by Ralstonia eutropha with LA for hydroxyvalerate (HV) production, which plays an important role in enhancing the thermal properties of PHBV. Accordingly, the HV content of PHBV varied from 0-40.9% at different concentrations of LA. Stimulation of cell growth and PHBV accumulation were observed when 2-6 g L(-1) LA was supplied to the culture. The optimal nitrogen sources were determined to be 0.5 g L(-1) ammonium chloride and 2 g L(-1) casein peptone. It was determined that the optimal pH for cell growth and PHBV accumulation was 7.0. When the cultivation was performed in large scale (2 L fermenter) with a low DO concentration of 30% and a pH of 7.0, a high maximum dry cell weight of 15.53 g L(-1) with a PHBV concentration of 12.61 g L(-1) (53.9% HV), up to 81.2% of the dry cell weight, was obtained. The melting point of PHBV found to be decreased as the fraction of HV present in the polymer increased, which resulted in an improvement in the ductility and flexibility of the polymer. The results of this study will improve the understanding of the PHBV accumulation and production by R. eutropha and will be valuable for the industrial production of biosynthesized polymers.

  9. Low temperature-induced viable but not culturable state of Ralstonia eutropha and its relationship to accumulated polyhydroxybutyrate

    PubMed Central

    Nowroth, Verena; Marquart, Lisa; Jendrossek, Dieter

    2016-01-01

    The culturability of Escherichia coli, Ralstonia eutropha and Bacillus subtilis after incubation in phosphate-buffered saline at either 5°C or 30°C was determined. The culturability of B. subtilis showed little dependence on temperature. The culturability of E. coli rapidly decreased at 30°C but remained almost constant at 5°C. In contrast, the culturability of R. eutropha decreased by three orders of magnitude at 5°C within 24 h but only moderately decreased (one order of magnitude) at 30°C. Remarkably, prolonged incubation of R. eutropha at 30°C resulted in a full recovery of colony forming units in contrast to only a partial recovery at 5°C. Ralstonia eutropha cells at 30°C remained culturable for 3 weeks while culturability at 5°C constantly decreased. The effect of temperature was significantly stronger in a polyhydroxybutyrate-negative mutant. Our data show that accumulated polyhydroxybutyrate has a cold-protective function and can prevent R. eutropha entering the viable but not culturable state. PMID:27810883

  10. Incorporation of Escherichia coli O157:H7 in biofilms with Ralstonia insidiosa, a primary localizer for the development of heterogeneous biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It is hypothesized that the presence of strong biofilm forming microflora could potentially enhance the survival of Escherichia coli O157:H7 (EcO157) in harsh environment. In this study, a strong biofilm forming bacterium, Ralstonia insidiosa, previously isolated from a fresh-cut produce plant was c...

  11. Modification of β-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil.

    PubMed

    Insomphun, Chayatip; Mifune, Jun; Orita, Izumi; Numata, Keiji; Nakamura, Satoshi; Fukui, Toshiaki

    2014-02-01

    Ralstonia eutropha H16 is a useful platform for metabolic engineering aiming at efficient production of polyhydroxyalkanaotes being attracted as practical bioplastics. This study focused on bifunctional (S)-specific 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase encoded by fadB to obtain information regarding β-oxidation in this bacterium and to achieve compositional regulation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] synthesized from soybean oil. In addition to two FadB homologs (FadB1 and FadB') encoded within the previously identified β-oxidation gene clusters on the chromosome 1, a gene of third homolog (FadB2) was found on chromosome 2 of R. eutropha. The fadB homologs were disrupted in R. eutropha strain NSDG expressing a mutant gene of PHA synthase from Aeromonas caviae. The gene disruptions affected neither growth nor PHA production on fructose. On soybean oil, fadB' deletion led to reduction of PHA quantity attributed to decrease of 3HB unit, while fadB1 deletion slightly increased 3HHx composition without serious negative impact on both cell growth and PHA biosynthesis. Double deletion of fadB1 and fadB' significantly impaired the cell growth and PHA biosynthesis, indicating the major roles of fadB1 and fadB' in β-oxidation. When fadB1 was deleted in several engineered strains of R. eutropha possessing additional (R)-enoyl-CoA hydratase gene(s), the net amounts of 3HHx unit in the PHA fractions showed 6-21% increase probably due to slightly enhanced supply of medium-chain-length 2-enoyl-CoAs through the partially impaired β-oxidation. These results demonstrated that modification of β-oxidation by fadB1 deletion was effective for increasing 3HHx composition in the copolyesters produced from soybean oil.

  12. Risk assessment of 'Candidatus Liberibacter solanacearum' transmission by the psyllids Bactericera trigonica and B. tremblayi from Apiaceae crops to potato.

    PubMed

    Antolinez, C A; Fereres, A; Moreno, A

    2017-04-03

    Candidatus Liberibacter solanacearum (Lso) is bacterium transmitted by psyllids to Solanaceae and Apiaceae plants. So far, Lso is found in Europe affecting Apiaceae. In the Mediterranean region, Bactericera trigonica is the only known vector of Lso, but the leek-onion psyllid Bactericera tremblayi is another widespread psyllid and potential vector of Lso. Commonly, carrot, leek and potato are cultivated in the same zones and it is uncertain if these psyllid species are able to transmit Lso to potato plants. Here, we assessed the transmission of Lso by B. trigonica and B. tremblayi to potato plants. B. trigonica showed preference to ingest from the phloem, settle and oviposit on carrot and celery but not on potato. This was correlated with high Lso transmission rates to both carrot (80%) and celery (70%) but very low to potato (≤3%). B. tremblayi preferred leek over carrot and potato, the latter being the less preferred host. B. tremblayi readily ingested from the phloem of infected carrots but failed to transmit Lso from carrot to carrot. Our study shows that the risk of Lso transmission from Apiaceae to potato by B. trigonica is very low, and that B. tremblayi is not a likely vector of Lso.

  13. Identification and characterization of chemosensors for d-malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum.

    PubMed

    Tunchai, Mattana; Hida, Akiko; Oku, Shota; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

    2017-02-01

    Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable d-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for d-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to d-malate, suggesting the existence of another d-malate chemosensor. McpM previously had been identified as a chemosensor for l-malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to d-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for d-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to l-tartrate and d-malate and moderately to d-tartrate, but not to l-malate or succinate. K18 harbouring mcpM responded strongly to l-malate and d-tartrate and moderately to succinate, fumarate and d-malate. Ps29 utilizes l-malate and l-tartrate, but not d-malate. We therefore concluded that l-tartrate and l-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward d-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild-type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection.

  14. Chemoselective Nitro Group Reduction and Reductive Dechlorination Initiate Degradation of 2-Chloro-5-Nitrophenol by Ralstonia eutropha JMP134

    PubMed Central

    Schenzle, Andreas; Lenke, Hiltrud; Spain, Jim C.; Knackmuss, Hans-Joachim

    1999-01-01

    Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene. PMID:10347008

  15. Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

    PubMed

    Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P

    2016-01-01

    The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.

  16. Chemoselective nitro group reduction and reductive dechlorination initiate degradation of 2-chloro-5-nitrophenol by Ralstonia eutropha JMP134

    SciTech Connect

    Schenzle, A.; Lenke, H.; Knackmuss, H.J.; Spain, J.C.

    1999-06-01

    Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134, 2-chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.

  17. Liquid Fuel From Bacteria: Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from CO2, Hydrogen, and Oxygen

    SciTech Connect

    2010-07-15

    Electrofuels Project: MIT is using solar-derived hydrogen and common soil bacteria called Ralstonia eutropha to turn carbon dioxide (CO2) directly into biofuel. This bacteria already has the natural ability to use hydrogen and CO2 for growth. MIT is engineering the bacteria to use hydrogen to convert CO2 directly into liquid transportation fuels. Hydrogen is a flammable gas, so the MIT team is building an innovative reactor system that will safely house the bacteria and gas mixture during the fuel-creation process. The system will pump in precise mixtures of hydrogen, oxygen, and CO2, and the online fuel-recovery system will continuously capture and remove the biofuel product.

  18. Comparative Genomics of Cultured and Uncultured Strains Suggests Genes Essential for Free-Living Growth of Liberibacter

    PubMed Central

    Fagen, Jennie R.; Leonard, Michael T.; McCullough, Connor M.; Edirisinghe, Janaka N.; Henry, Christopher S.; Davis, Michael J.; Triplett, Eric W.

    2014-01-01

    The full genomes of two uncultured plant pathogenic Liberibacter, Ca. Liberibacter asiaticus and Ca. Liberibacter solanacearum, are publicly available. Recently, the larger genome of a closely related cultured strain, Liberibacter crescens BT-1, was described. To gain insights into our current inability to culture most Liberibacter, a comparative genomics analysis was done based on the RAST, KEGG, and manual annotations of these three organisms. In addition, pathogenicity genes were examined in all three bacteria. Key deficiencies were identified in Ca. L. asiaticus and Ca. L. solanacearum that might suggest why these organisms have not yet been cultured. Over 100 genes involved in amino acid and vitamin synthesis were annotated exclusively in L. crescens BT-1. However, none of these deficiencies are limiting in the rich media used to date. Other genes exclusive to L. crescens BT-1 include those involved in cell division, the stringent response regulatory pathway, and multiple two component regulatory systems. These results indicate that L. crescens is capable of growth under a much wider range of conditions than the uncultured Liberibacter strains. No outstanding differences were noted in pathogenicity-associated systems, suggesting that L. crescens BT-1 may be a plant pathogen on an as yet unidentified host. PMID:24416233

  19. Effect of clove oil on plant pathogenic bacteria and bacterial wilt of tomato and geranium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We determined the antibacterial activity of clove oil against seven different genera of plant pathogenic bacteria including Gram-negative Agrobacterium tumefaciens, Erwinia carotovora pv. carotovora, Pseudomonas syringae pv. syringae, Ralstonia solanacearum, and Xanthomonas campestris pv. pelargonii...

  20. Isolation of Bacillus amyloliquefaciens S20 and its application in control of eggplant bacterial wilt.

    PubMed

    Chen, Da; Liu, Xin; Li, Chunyu; Tian, Wei; Shen, Qirong; Shen, Biao

    2014-05-01

    Bacterial strain S20 was isolated and identified as Bacillus amyloliquefaciens based on physiological and biochemical characteristics and a 16S rRNA gene sequence analysis. Strain S20 inhibits the growth of Fusarium oxysporum and Ralstonia solanacearum. Some genes associated with the synthesis of some lipopeptides were detected in strain S20 by PCR. Iturins A were identified as the main antagonistic substrates by analysis with electrospray ionization mass spectrometry/collision-induced dissociation (ESI-MS/CID). Four homologues of iturin A (C13-C16) were identified. Pot experiments showed that the application of strain S20 alone could control eggplant wilt with an efficacy of 25.3% during a 40 day experiment. If strain S20 was used with organic fertilizer, the control efficacy against eggplant wilt reached as high as 70.7%. The application of organic fertilizer alone promotes the growth of R. solanacearum, resulting in a higher wilt incidence than that observed in control plants. The application of strain S20 effectively inhibits R. solanacearum in the rhizosphere soil of eggplant. The combined use of strain S20 and organic fertilizer more effectively controlled R. solanacearum in soil than the use of strain S20 alone. The soil count of strain S20 decreased gradually during the course of the experiment after inoculation. Organic fertilizer was beneficial for the survival of the antagonistic bacterial strain S20; a higher level of these bacteria could be maintained. The application of organic fertilizer with strain S20 increased bacterial diversity in rhizosphere soil.

  1. Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

    PubMed

    Li, Wenbin; Abad, Jorge A; French-Monar, Ronald D; Rascoe, John; Wen, Aimin; Gudmestad, Neil C; Secor, Gary A; Lee, Ing-Ming; Duan, Yongping; Levy, Laurene

    2009-07-01

    The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated

  2. Tracking and identification of antibacterial components in the essential oil of Tanacetum vulgare L. by the combination of high-performance thin-layer chromatography with direct bioautography and mass spectrometry.

    PubMed

    Móricz, Ágnes M; Häbe, Tim T; Böszörményi, Andrea; Ott, Péter G; Morlock, Gertrud E

    2015-11-27

    Two tansy (Tanacetum vulgare L.) essential oils were obtained by steam distillation of the capitula with subsequent liquid-liquid extraction (oil 1) or with use of an auxiliary phase for the trapping of the steam components (oil 2). These oils were investigated against Bacillus subtilis F1276, B. subtilis spizizenii (DSM 618), Xanthomonas euvesicatoria, Pseudomonas syringae pv. maculicola, Ralstonia solanacearum strain GMI1000 and Aliivibrio fischeri, using the coupling of high-performance thin-layer chromatography to direct bioautography (HPTLC-DB). Using this method with the potato and tomato pathogen R. solanacearum is shown for the first time. Due to the advanced extraction process, oil 2 was richer in components and provided more inhibition zones. The main bioactive components were identified by scanning HPTLC-Direct Analysis in Real Time mass spectrometry (HPTLC-DART-MS) and solid-phase microextraction gas chromatography electron impact MS (SPME-GC-EI-MS) as cis- and trans-chrysanthenol as well as trans-chrysanthenyl acetate. cis-Chrysanthenol exhibited antibacterial effects against all tested bacteria, whereas trans-chrysanthenol inhibited B. subtilis, R. solanacearum and A. fischeri. trans-Chrysanthenyl acetate was an inhibitor for X. euvesicatoria, R. solanacearum and A. fischeri. Although HPTLC-DART-MS resulted in a comparable fragmentation, the ionization characteristics and the recorded mass spectra clearly showed that DART is a softer ionization technique than EI. It is also more affected by ambient conditions and thus prone to additional oxidation products.

  3. Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

    SciTech Connect

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Kim, Sangwoo; Ahn, Jae-Woo; Kim, Kyung-Jin

    2014-02-14

    Highlights: • We determined a crystal structure of β-keto thiolase from Ralstonia eutropha H16 (ReBktB). • Distinct substrate binding mode ReBktB was elucidated. • Enzymatic kinetic parameters of ReBktB were revealed. - Abstract: ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K{sub m}, V{sub max}, and K{sub cat} values of 11.58 μM, 1.5 μmol/min, and 102.18 s{sup −1}, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.

  4. Identification of Strains of Alcaligenes and Agrobacterium by a Polyphasic Approach

    PubMed Central

    Clermont, Dominique; Harmant, Christine; Bizet, Chantal

    2001-01-01

    The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of A. xylosoxidans to be clearly separated. Relationships between five strains and the Ralstonia paucula type strain were demonstrated. Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A. xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing. PMID:11526136

  5. Natural Strain

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.

    1995-01-01

    The purpose of this paper is to present a consistent and thorough development of the strain and strain-rate measures affiliated with Hencky. Natural measures for strain and strain-rate, as I refer to them, are first expressed in terms of of the fundamental body-metric tensors of Lodge. These strain and strain-rate measures are mixed tensor fields. They are mapped from the body to space in both the Eulerian and Lagrangian configurations, and then transformed from general to Cartesian fields. There they are compared with the various strain and strain-rate measures found in the literature. A simple Cartesian description for Hencky strain-rate in the Lagrangian state is obtained.

  6. Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha*

    PubMed Central

    Niks, Dimitri; Duvvuru, Jayant; Escalona, Miguel; Hille, Russ

    2016-01-01

    We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD+-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its MoV state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center. PMID:26553877

  7. Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha.

    PubMed

    Niks, Dimitri; Duvvuru, Jayant; Escalona, Miguel; Hille, Russ

    2016-01-15

    We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+)-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.

  8. Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products.

    PubMed

    Riedel, Sebastian L; Lu, Jingnan; Stahl, Ulf; Brigham, Christopher J

    2014-02-01

    Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.

  9. Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels.

    PubMed

    Yu, J; Wang, J

    2001-06-20

    Ralstonia eutropha grows on and produces polyhydroxyalkanoates (PHAs) from fermentation acids. Acetic acid, one major organic acid from acidogenesis of organic wastes, has an inhibitory effect on the bacterium at slightly alkaline pH (6 g HAc/L at pH 8). The tolerance of R. eutropha to acetate, however, was increased significantly up to 15 g/L at the slightly alkaline pH level with high cell mass concentration. A metabolic cell model with five fluxes is proposed to depict the detoxification mechanism including mass transfer and acetyl-CoA formation of acetic acid and the formation of three final metabolic products, polyhydroxybutyrate (PHB), active biomass, and CO(2). The fluxes were measured under different conditions such as cell mass concentration, acetic acid concentration, and medium composition. The experimental results indicate that the acetate detoxification by high cell mass concentration is attributed to the increased fluxes at high extracellular acetate concentrations. The fluxes could be doubled to reduce and hence detoxify the accumulated intracellular acetate anions.

  10. Characterization of poly-3-hydroxybutyrate (PHB) produced from Ralstonia eutropha using an alkali-pretreated biomass feedstock.

    PubMed

    Saratale, Ganesh D; Oh, Min-Kyu

    2015-09-01

    Alkaline pretreatment using NaOH, KOH, or NaOCl has been applied to various types of waste biomass to enhance enzymatic digestibility. Pretreatment (2% NaOH, 121 °C, 30 min) of rice paddy straw (PS) resulted in a maximum yield of 703 mg of reducing sugar per gram of PS with 84.19% hydrolysis yield after a two-step enzymatic hydrolysis process. Ralstonia eutropha ATCC 17699 was tested for its ability to synthesize poly-3-hydroxybutyrate (PHB) using PS hydrolysates as its sole carbon source. It is noteworthy that dry cell weight, polyhydroxyalkanoate (PHA) accumulation and PHB yield with the use of laboratory-grade sugars were similar to those achieved with PS-derived sugars. Under optimized conditions, we observed maximal PHA accumulation (75.45%) and PHB production (11.42 g/L) within 48 h of fermentation. After PHB recovery, the physicochemical properties of PHB were determined by various analytical techniques, showed the results were consistent with the characteristics of a standard polymer of PHB. Thus, the PS hydrolysate proved to be an excellent cheap carbon substrate for PHB production.

  11. vsrB, a regulator of virulence genes of Pseudomonas solanacearum, is homologous to sensors of the two-component regulator family.

    PubMed Central

    Huang, J; Denny, T P; Schell, M A

    1993-01-01

    Pseudomonas solanacearum, an important wilt pathogen of many plants, produces several extracellular proteins (EXPs) and extracellular polysaccharides (EPSs) that contribute to its virulence. Using TnphoA mutagenesis, we discovered a new gene, vsrB, that when inactivated causes a major reduction in the virulence and production of an EPS. Analysis of eps::lacZ reporters showed that vsrB is required for maximal expression (transcription) of eps, whose products are required for production of EPS I, a major virulence determinant. Analysis of EXPs in culture supernatants revealed that inactivation of vsrB also causes reduced production of two major EXPs, with molecular masses of 28 and 97 kDa, and a simultaneous 15-fold increase in levels of another EXP, PglA endopolygalacturonase. The vsrB gene was cloned from a P. solanacearum genomic library by complementation of the nonmucoid phenotype of the vsrB::TnphoA mutant and then subcloned on a 2.4-kb DNA fragment. TnphoA fusion analysis and subcellular localization of the vsrB gene product in Escherichia coli maxicells suggest that it is a ca. 60-kDa transmembrane protein. The nucleotide sequence of the 2.4-kb DNA fragment was determined, and a 638-amino-acid open reading frame was found for VsrB. A search of the GenBank data base found that the central part of VsrB has homology with the histidine kinase domain of sensors in the two-component regulator family, while the C terminus has homology with the phosphate receiver domain of response regulators in the same family. Genetic analysis suggests that the receiver domain is not required for vsrB function. Images PMID:8407789

  12. Application of nitroarene dioxygenases in the design of novel strains that degrade chloronitrobenzenes

    PubMed Central

    Ju, Kou‐San; Parales, Rebecca E.

    2009-01-01

    Summary Widespread application of chloronitrobenzenes as feedstocks for the production of industrial chemicals and pharmaceuticals has resulted in extensive environmental contamination with these toxic compounds, where they pose significant risks to the health of humans and wildlife. While biotreatment in general is an attractive solution for remediation, its effectiveness is limited with chloronitrobenzenes due to the small number of strains that can effectively mineralize these compounds and their ability to degrade only select isomers. To address this need, we created engineered strains with a novel degradation pathway that reduces the total number of steps required to convert chloronitrobenzenes into compounds of central metabolism. We examined the ability of 2‐nitrotoluene 2,3‐dioxygenase from Acidovorax sp. strain JS42, nitrobenzene 1,2‐dioxygenase (NBDO) from Comamonas sp. strain JS765, as well as active‐site mutants of NBDO to generate chlorocatechols from chloronitrobenzenes, and identified the most efficient enzymes. Introduction of the wild‐type NBDO and the F293Q variant into Ralstonia sp. strain JS705, a strain carrying the modified ortho pathway for chlorocatechol metabolism, resulted in bacterial strains that were able to sustainably grow on all three chloronitrobenzene isomers without addition of co‐substrates or co‐inducers. These first‐generation engineered strains demonstrate the utility of nitroarene dioxygenases in expanding the metabolic capabilities of bacteria and provide new options for improved biotreatment of chloronitrobenzene‐contaminated sites. PMID:21261918

  13. Catalytic Properties of the Isolated Diaphorase Fragment of the NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha

    PubMed Central

    Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A.; Lenz, Oliver

    2011-01-01

    The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with KI values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis. PMID:22016788

  14. Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.

    PubMed

    Karstens, Katja; Zschiedrich, Christopher P; Bowien, Botho; Stülke, Jörg; Görke, Boris

    2014-04-01

    EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the β-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha.

  15. Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

    PubMed

    Sznajder, Anna; Pfeiffer, Daniel; Jendrossek, Dieter

    2015-03-01

    Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

  16. PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.

    PubMed

    Pfeiffer, Daniel; Jendrossek, Dieter

    2014-01-01

    Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

  17. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut processing plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been ...

  18. Efficient biological conversion of carbon monoxide (CO) to carbon dioxide (CO2) and for utilization in bioplastic production by Ralstonia eutropha through the display of an enzyme complex on the cell surface.

    PubMed

    Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-06-25

    An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of the CO2-utilizing Ralstonia eutropha and successfully resulted in a 3.3-fold increase in conversion efficiency. These results suggest that this complexed system may be a promising strategy for CO2 utilization as a biological tool for the production of bioplastics.

  19. Evolution of a Chlorobenzene Degradative Pathway Among Bacteria in a Contaminated Groundwater Mediated by a Genomic Island in Ralstonia

    DTIC Science & Technology

    2003-01-01

    In strain JS705 were a mosaic of the etc genes, previously described for Pseudomonas sp. strain 813, and a 5 kb fragment identical to strain JS745...amplification, and deampllflcation in Pseudomonas putlda F1 of a 1 05-kilobase genetic ele- ment containing the chlorocatechol degradative genes from... Pseudomonas sp. strain 813. J Bactertol 180: 4360--4369). The unique reconstruction of forma- tion of a metabolic pathway through the activity of IS elements

  20. Localization of poly(3-hydroxybutyrate) (PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16.

    PubMed

    Pfeiffer, Daniel; Jendrossek, Dieter

    2012-11-01

    Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins

  1. Strain Gage

    NASA Technical Reports Server (NTRS)

    1995-01-01

    HITEC Corporation developed a strain gage application for DanteII, a mobile robot developed for NASA. The gage measured bending forces on the robot's legs and warned human controllers when acceptable forces were exceeded. HITEC further developed the technology for strain gage services in creating transducers out of "Indy" racing car suspension pushrods, NASCAR suspension components and components used in motion control.

  2. New Insights in PhaM-PhaC-mediated Localization of PHB Granules in Ralstonia eutropha H16.

    PubMed

    Bresan, Stephanie; Jendrossek, Dieter

    2017-04-07

    Formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha is controlled by PhaM that interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance to attach PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in a detachment of formed PHB granules from the nucleoid. Instead, a formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells was observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct co-localization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaC(C319A) proteins were not able to form fluorescent foci indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach at later stages from formed PHB granules resulting in an inhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB permissive conditions at a certain size.Importance PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells and for association of formed PHB granules to the nucleoid. Here we found out that four lysine residues of C-terminal PhaM sequence motifs are essential for association of PHB granules to

  3. Role of the two-component leader sequence and mature amino acid sequences in extracellular export of endoglucanase EGL from Pseudomonas solanacearum.

    PubMed Central

    Huang, J Z; Schell, M A

    1992-01-01

    The egl gene of Pseudomonas solanacearum encodes a 43-kDa extracellular endoglucanase (mEGL) involved in wilt disease caused by this phytopathogen. Egl is initially translated with a 45-residue, two-part leader sequence. The first 19 residues are apparently removed by signal peptidase II during export of Egl across the inner membrane (IM); the remaining residues of the leader sequence (modified with palmitate) are removed during export across the outer membrane (OM). Localization of Egl-PhoA fusion proteins showed that the first 26 residues of the Egl leader sequence are required and sufficient to direct lipid modification, processing, and export of Egl or PhoA across the IM but not the OM. Fusions of the complete 45-residue leader sequence or of the leader and increasing portions of mEgl sequences to PhoA did not cause its export across the OM. In-frame deletion of portions of mEGL-coding sequences blocked export of the truncated polypeptides across the OM without affecting export across the IM. These results indicate that the first part of the leader sequence functions independently to direct export of Egl across the IM while the second part and sequences and structures in mEGL are involved in export across the OM. Computer analysis of the mEgl amino acid sequence obtained from its nucleotide sequence identified a region of mEGL similar in amino acid sequence to regions in other prokaryotic endoglucanases. Images PMID:1735723

  4. New report of additional enterobacterial species causing wilt in West Bengal, India.

    PubMed

    Sarkar, Shamayeeta; Chaudhuri, Sujata

    2015-07-01

    Ralstonia solanacearum is known to be the most prominent causal agent of bacterial wilt worldwide. It has a wide host range comprising solanaceous and nonsolanaceous plants. Typical symptoms of the disease are leaf wilt, browning of vascular tissues, and collapsing of the plant. With the objective of studying the diversity of pathogens causing bacterial wilt in West Bengal, we collected samples of diseased symptomatic crops and adjacent symptomatic and asymptomatic weeds from widespread locations in West Bengal. By means of a routine molecular identification test specific to "R. solanacearum species complex", the majority of these strains (68 out of 71) were found to not be R. solanacearum. Presumptive identification of these isolates with conventional biochemicals, extensive testing of pathogenicity of a subset involving greenhouse trials fulfilling Koch's postulate test, and scanning electron microscopic analysis for the presence of pathogen in diseased plants were done. 16S rDNA sequencing of a subset of these strains (GenBank accession Nos. JX880249-JX880251) and analysis of sequences with the nBLAST programme showed a high similarity (97%-99%) to sequences of the Enterobacteriaceae group available in GenBank. Molecular phylogeny further established the taxonomic position of the strains. The 3 bacterial strain cultures have been submitted to MTCC, Institute of Microbial Technology, Chandigarh, India, and were identified as Klebsiella oxytoca, Enterobacter cowanii, and Klebsiella oxytoca, respectively. Although Enterobacter sp. has previously been reported to cause wilt in many plants, susceptibility of most of the dedicated hosts of R. solanacearum to wilt caused by Enterobacter and other bacteria from Enterobacteriaceae is being reported for the first time in this work.

  5. Poly(3-Hydroxybutyrate) Synthesis Genes in Azotobacter sp. Strain FA8

    PubMed Central

    Pettinari, M. Julia; Vázquez, Gustavo J.; Silberschmidt, Daniel; Rehm, Bernd; Steinbüchel, Alexander; Méndez, Beatriz S.

    2001-01-01

    Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for β-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB. PMID:11679365

  6. Use of Electrical Penetration Graph Technology to Examine Transmission of 'Candidatus Liberibacter solanacearum' to Potato by Three Haplotypes of Potato Psyllid (Bactericera cockerelli; Hemiptera: Triozidae).

    PubMed

    Mustafa, Tariq; Horton, David R; Cooper, W Rodney; Swisher, Kylie D; Zack, Richard S; Pappu, Hanu R; Munyaneza, Joseph E

    2015-01-01

    The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium 'Candidatus Liberibacter solanacearum' (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid (Central, Western, Northwestern). All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato ("inoculation access period", or IAP) to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions

  7. Classification of Plant Associated Bacteria Using RIF, a Computationally Derived DNA Marker

    PubMed Central

    Schneider, Kevin L.; Marrero, Glorimar; Alvarez, Anne M.; Presting, Gernot G.

    2011-01-01

    A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained

  8. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.

    PubMed

    Matsumoto, Ken'ichiro; Tanaka, Yoshikazu; Watanabe, Tsuyoshi; Motohashi, Ren; Ikeda, Koji; Tobitani, Kota; Yao, Min; Tanaka, Isao; Taguchi, Seiichi

    2013-10-01

    NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

  9. Expression and activity of the Calvin-Benson-Bassham cycle transcriptional regulator CbbR from Acidithiobacillus ferrooxidans in Ralstonia eutropha.

    PubMed

    Esparza, Mario; Jedlicki, Eugenia; Dopson, Mark; Holmes, David S

    2015-08-01

    Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbR(AF)) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbR(AF) function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbR(AF) was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbR(AF) was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbR(AF) activity.

  10. Versatile plasmid-based expression systems for Gram-negative bacteria--General essentials exemplified with the bacterium Ralstonia eutropha H16.

    PubMed

    Gruber, Steffen; Schwab, Helmut; Koefinger, Petra

    2015-12-25

    The Gram-negative bacterium Escherichia coli is currently the most efficient and widely used prokaryotic host for recombinant protein and metabolite production. However, due to some limitations and to various interesting features of other Gram-negative bacteria efficient vector systems applicable to a broad range are desired. Basic building blocks for plasmid-based vectors include besides the need for a suitable selection marker in the first line a proper replication and maintenance system. In addition to these basic requirements, further elements are needed for Gram-negative bacteria beyond E. coli, such as Pseudomonas pudita, Ralstonia eutropha, Burkholderia glumae or Acinetobacter sp.. Established building blocks have to be adapted and new building blocks providing the desired functions need to be identified and exploited. This minireview addresses so far described and used genetic elements for broad host range replication, efficient plasmid maintenance, and conjugative plasmid transfer as well as expression elements and protein secretion signals. The industrially important bacterium R. eutropha H16 was chosen as a model organism to provide specific data on the effectivity and utility of building blocks based on such genetic elements.

  11. Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

    SciTech Connect

    Brigham, CJ; Speth, DR; Rha, C; Sinskey, AJ

    2012-10-22

    Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor sigma(54) increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

  12. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha H16

    PubMed Central

    Kim, Jieun; Kim, Kyung-Jin

    2014-01-01

    The (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha (RePaaH1) is an enzyme used in the biosynthesis of n-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to (S)-3-hydroxybutyryl-CoA. The RePaaH1 protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 1.4 M ammonium sulfate, 0.1 M sodium cacodylate pH 6.0, 0.2 M sodium chloride at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.6 Å on a synchrotron beamline. The crystal belonged to space group P3221, with unit-cell parameters a = b = 135.4, c = 97.2 Å. With three molecules per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.68 Å3 Da−1, which corresponds to a solvent content of approximately 54.1%. The structure was solved by the single-wavelength anomalous dispersion method and refinement of the structure is in progress. PMID:25005097

  13. High temperature strain gage apparent strain compensation

    NASA Technical Reports Server (NTRS)

    Holmes, Harlan K.; Moore, T. C., Sr.

    1992-01-01

    Once an installed strain gage is connected to a strain indicating device and the instrument is balanced, a subsequent change in temperature of the gage installation will generally produce a resistance change in the gage. This purely temperature-induced resistance will be registered by the indicating device as a strain and is referred to as 'apparent strain' to distinguish it from strain due to applied stress. One desirable technique for apparent strain compensation is to employ two identical gages with identical mounting procedures which are connected with a 'half bridge' configuration where gages see the same thermal environment but only one experiences a mechanical strain input. Their connection in adjacent arms of the bridge will then balance the thermally induced apparent strains and, in principle, only the mechanical strain remains. Two approaches that implement this technique are discussed.

  14. Isolation and characterization of a plasmid DNA from periodontopathogenic bacterium, Eikenella corrodens 1073, which affects pilus formation and colony morphology.

    PubMed

    Azakami, Hiroyuki; Akimichi, Hiromi; Usui, Masakatsu; Yumoto, Hiromichi; Ebisu, Shigeyuki; Kato, Akio

    2005-05-23

    Eikenella corrodens (Ec) is one of a group of periodontopathogenic bacteria. A plasmid DNA (8.7 kb) isolated from Ec 1073 was designated pMU1. Agarose gel electrophoresis and Southern analysis suggested that pMU1-like plasmids were carried in 2 Ec strains, including 1073, with higher hemagglutination (HA) activity than other strains. We determined the nucleotide sequence of this plasmid and identified 7 ORFs. A homology search revealed that 4 ORFs of pMU1 were homologous to ORFs in pJTPS1, found in a spontaneous avirulent mutant of the phytopathogenic bacterium, Ralstonia solanacearum. pJTPS1 is a putative hypovirulent plasmid, which is thought to control the virulence of R. solanacearum. We also found the ORF to be homologous to the recombinase specific to the type IV pilin gene. We introduced a part of pMU1 into the Ec 23834 strain, which has a pilus structure on its cell surface and forms corroding colonies on solid medium. No pilus structure was observed on the surface of transformants, most of which formed non-corroding colonies. When such transformants (or Ec 1073) were cured of pMU1 with acridine orange, they remained non-foliated and non-corroding. The results suggest that pMU1 might irreversibly affect pilus formation and colony morphology, and might be involved in the pathogenicity and virulence of Ec.

  15. Diversity, Biocontrol, and Plant Growth Promoting Abilities of Xylem Residing Bacteria from Solanaceous Crops

    PubMed Central

    Achari, Gauri A.

    2014-01-01

    Eggplant (Solanum melongena L.) is one of the solanaceous crops of economic and cultural importance and is widely cultivated in the state of Goa, India. Eggplant cultivation is severely affected by bacterial wilt caused by Ralstonia solanacearum that colonizes the xylem tissue. In this study, 167 bacteria were isolated from the xylem of healthy eggplant, chilli, and Solanum torvum Sw. by vacuum infiltration and maceration. Amplified rDNA restriction analysis (ARDRA) grouped these xylem residing bacteria (XRB) into 38 haplotypes. Twenty-eight strains inhibited growth of R. solanacearum and produced volatile and diffusible antagonistic compounds and plant growth promoting substances in vitro. Antagonistic strains XB86, XB169, XB177, and XB200 recorded a biocontrol efficacy greater than 85% against BW and exhibited 12%–22 % increase in shoot length in eggplant in the greenhouse screening. 16S rRNA based identification revealed the presence of 23 different bacterial genera. XRB with high biocontrol and plant growth promoting activities were identified as strains of Staphylococcus sp., Bacillus sp., Streptomyces sp., Enterobacter sp., and Agrobacterium sp. This study is the first report on identity of bacteria from the xylem of solanaceous crops having traits useful in cultivation of eggplant. PMID:24963298

  16. Geobacteraceae strains and methods

    DOEpatents

    Lovley, Derek R.; Nevin, Kelly P.; Yi, Hana

    2015-07-07

    Embodiments of the present invention provide a method of producing genetically modified strains of electricigenic microbes that are specifically adapted for the production of electrical current in microbial fuel cells, as well as strains produced by such methods and fuel cells using such strains. In preferred embodiments, the present invention provides genetically modified strains of Geobacter sulfurreducens and methods of using such strains.

  17. Muscle strain treatment

    MedlinePlus

    Treatment - muscle strain ... Question: How do you treat a muscle strain ? Answer: Rest the strained muscle and apply ice for the first few days after the injury. Anti-inflammatory medicines or acetaminophen ( ...

  18. Muscle strain (image)

    MedlinePlus

    A muscle strain is the stretching or tearing of muscle fibers. A muscle strain can be caused by sports, exercise, a ... something that is too heavy. Symptoms of a muscle strain include pain, tightness, swelling, tenderness, and the ...

  19. Cometabolic degradation of ethyl mercaptan by phenol-utilizing Ralstonia eutropha in suspended growth and gas-recycling trickle-bed reactor.

    PubMed

    Sedighi, Mahsa; Zamir, Seyed Morteza; Vahabzadeh, Farzaneh

    2016-01-01

    The degradability of ethyl mercaptan (EM), by phenol-utilizing cells of Ralstonia eutropha, in both suspended and immobilized culture systems, was investigated in the present study. Free-cells experiments conducted at EM concentrations ranging from 1.25 to 14.42 mg/l, showed almost complete removal of EM at concentrations below 10.08 mg/l, which is much higher than the maximum biodegradable EM concentration obtained in experiments that did not utilize phenol as the primary substrate, i.e. 2.5 mg/l. The first-order kinetic rate constant (kSKS) for EM biodegradation by the phenol-utilizing cells (1.7 l/g biomass/h) was about 10 times higher than by cells without phenol utilization. Immobilized-cells experiments performed in a gas recycling trickle-bed reactor packed with kissiris particles at EM concentrations ranging from 1.6 to 36.9 mg/l, showed complete removal at all tested concentrations in a much shorter time, compared with free cells. The first-order kinetic rate constant (rmaxKs) for EM utilization was 0.04 l/h for the immobilized system compared to 0.06 for the suspended-growth culture, due to external mass transfer diffusion. Diffusion limitation was decreased by increasing the recycling-liquid flow rate from 25 to 65 ml/min. The removed EM was almost completely mineralized according to TOC and sulfate measurements. Shut down and starvation experiments revealed that the reactor could effectively handle the starving conditions and was reliable for full-scale application.

  20. Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16.

    PubMed

    Buhrke, Thorsten; Löscher, Simone; Lenz, Oliver; Schlodder, Eberhard; Zebger, Ingo; Andersen, Lars K; Hildebrandt, Peter; Meyer-Klaucke, Wolfram; Dau, Holger; Friedrich, Bärbel; Haumann, Michael

    2005-05-20

    The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

  1. Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism.

    PubMed

    Jia, Y; Yuan, W; Wodzinska, J; Park, C; Sinskey, A J; Stubbe, J

    2001-01-30

    The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.

  2. Effects of mutations in the substrate-binding domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 on PHB degradation.

    PubMed

    Hiraishi, Tomohiro; Hirahara, Yoko; Doi, Yoshiharu; Maeda, Mizuo; Taguchi, Seiichi

    2006-11-01

    Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ(RpiT1)) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ(RpiT1) was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ(RpiT1). Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ(RpiT1) adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.

  3. Hrp mutant bacteria as biocontrol agents: toward a sustainable approach in the fight against plant pathogenic bacteria.

    PubMed

    Hanemian, Mathieu; Zhou, Binbin; Deslandes, Laurent; Marco, Yves; Trémousaygue, Dominique

    2013-10-01

    Sustainable agriculture necessitates development of environmentally safe methods to protect plants against pathogens. Among these methods, application of biocontrol agents has been efficiently used to minimize disease development. Here we review current understanding of mechanisms involved in biocontrol of the main Gram-phytopathogenic bacteria-induced diseases by plant inoculation with strains mutated in hrp (hypersensitive response and pathogenicity) genes. These mutants are able to penetrate plant tissues and to stimulate basal resistance of plants. Novel protection mechanisms involving the phytohormone abscisic acid appear to play key roles in the biocontrol of wilt disease induced by Ralstonia solanacearum in Arabidopsis thaliana. Fully understanding these mechanisms and extending the studies to other pathosystems are still required to evaluate their importance in disease protection.

  4. New polyphenols from a deep sea Spiromastix sp. Fungus, and their antibacterial activities.

    PubMed

    Niu, Siwen; Liu, Dong; Proksch, Peter; Shao, Zongze; Lin, Wenhan

    2015-04-22

    Eleven new polyphenols namely spiromastols A-K (1-11) were isolated from the fermentation broth of a deep sea-derived fungus Spiromastix sp. MCCC 3A00308. Their structures were determined by extensive NMR data and mass spectroscopic analysis in association with chemical conversion. The structures are classified as diphenyl ethers, diphenyl esters and isocoumarin derivatives, while the n-propyl group in the analogues is rarely found in natural products. Compounds 1-3 exhibited potent inhibitory effects against a panel of bacterial strains, including Xanthomanes vesicatoria, Pseudomonas lachrymans, Agrobacterium tumefaciens, Ralstonia solanacearum, Bacillus thuringensis, Staphylococcus aureus and Bacillus subtilis, with minimal inhibitory concentration (MIC) values ranging from 0.25 to 4 µg/mL. The structure-activity relationships are discussed, while the polychlorinated analogues 1-3 are assumed to be a promising structural model for further development as antibacterial agents.

  5. Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed-batch and continuous cultures.

    PubMed

    Grunwald, Stephan; Mottet, Alexis; Grousseau, Estelle; Plassmeier, Jens K; Popović, Milan K; Uribelarrea, Jean-Louis; Gorret, Nathalie; Guillouet, Stéphane E; Sinskey, Anthony

    2015-01-01

    Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin-Benson-Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole(-1) in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (μmax  = 0.18 h(-1) ) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l(-1) .

  6. Effect of three organophosphorous nematicides on non-target nematodes and soil microbial community.

    PubMed

    Wada, Satoko; Toyota, Koki

    2008-01-01

    The toxicity of three organophosphorous nematicides, imicyafos, fosthiazate and cadusafos, to non-target organisms in soil was evaluated. Imicyafos and fosthiazate had no significant inhibitory effect on the growth of fungal (Fusarium oxysporum f. sp. lactucae, Rhizoctonia solani and Trichoderma viride) and bacterial (Ralstonia solanacearum and Pseudomonas fluorescens) strains in media at 12.5 to 200 mg L(-1). Cadusafos, however, significantly inhibited the growth of all these strains except R. solanacearum. A pot test was conducted using a soil naturally infested with Pratylenchus penetrans, and treated with imicyafos or fosthiazate, which are less toxic to non-target organisms. The density of P. penetrans decreased to less than 10% of the control level after exposure to imicyafos and fosthiazate at 3 kg active ingredient ha(-1), the conventional dose. No significant effect was observed on the density of free-living nematodes, cellulose decomposition activity, microbial biomass evaluated with the ATP method and number of ammonia oxidizers between the soil treated with imicyafos or fosthiazate and the untreated control soil. Our results revealed that imicyafos and fosthiazate effectively suppressed a plant-parasitic nematode, P. penetrans, but had little impact on free-living nematodes and the soil microbial community.

  7. Biosynthesis of poly(3-hydroxypropionate) from glycerol using engineered Klebsiella pneumoniae strain without vitamin B12

    PubMed Central

    Feng, Xinjun; Xian, Mo; Liu, Wei; Xu, Chao; Zhang, Haibo; Zhao, Guang

    2015-01-01

    Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. Previous studies demonstrated that engineered Escherichia coli strains can produce P3HP with supplementation of expensive vitamin B12. The present study examined the production of P3HP from glycerol in the recombinant Klebsiella pneumoniae strain, which naturally synthesizes vitamin B12. The genes glycerol dehydratase and its reactivation factor (dhaB123, gdrA, and gdrB from K. pneumoniae), aldehyde dehydrogenase (aldH from E. coli) were cloned and expressed in K. pneumoniae to produce 3-hydroxypropionate (3HP), with 2 genes (dhaT and yqhD) for biosynthesis of 1,3-propanediol were deleted. To obtain P3HP production, propionyl-CoA synthetase (prpE from E. coli) and polyhydroxyalkanoate synthase (phaC from Ralstonia eutropha) were introduced. Under the appropriate aeration condition, the cell yield and P3HP content were 0.24 g/L and 12.7% (wt/wt [cell dry weight]) respectively along with 2.03 g/L 3HP after 48 h cultivation. Although the yield is relatively low, this study shows the feasibility of producing P3HP in K. pneumoniae from glycerol without vitamin B12 for the first time. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of P3HP. PMID:25621933

  8. Superlattice strain gage

    DOEpatents

    Noel, B.W.; Smith, D.L.; Sinha, D.N.

    1988-06-28

    A strain gage comprising a strained-layer superlattice crystal exhibiting piezoelectric properties is described. A substrate upon which such a strained-layer superlattice crystal has been deposited is attached to an element to be monitored for strain. A light source is focused on the superlattice crystal and the light reflected from, passed through, or emitted from the crystal is gathered and compared with previously obtained optical property data to determine the strain in the element. 8 figs.

  9. Superlattice strain gage

    DOEpatents

    Noel, Bruce W.; Smith, Darryl L.; Sinha, Dipen N.

    1990-01-01

    A strain gage comprising a strained-layer superlattice crystal exhibiting piezoelectric properties is described. A substrate upon which such a strained-layer superlattice crystal has been deposited is attached to an element to be monitored for strain. A light source is focused on the superlattice crystal and the light reflected from, passed through, or emitted from the crystal is gathered and compared with previously obtained optical property data to determine the strain in the element.

  10. Structure and function of the N-terminal domain of Ralstonia eutropha polyhydroxyalkanoate synthase, and the proposed structure and mechanisms of the whole enzyme.

    PubMed

    Kim, Yeo-Jin; Choi, So Young; Kim, Jieun; Jin, Kyeong Sik; Lee, Sang Yup; Kim, Kyung-Jin

    2017-01-01

    Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by numerous microorganisms as energy and reducing power storage materials, and have attracted much attention as substitutes for petroleum-based plastics. In an accompanying paper, the authors reported the crystal structure of the C-terminal domain of Ralstonia eutropha PHA synthase (PhaC1). Here, the authors report the 3D reconstructed model of full-length of R. eutropha PhaC1 (RePhaC1F ) by small angle X-ray scattering (SAXS) analysis. The catalytic C-terminal domain of RePhaC1 (RePhaC1CD ) dimer is located at the center of RePhaC1F , and the N-terminal domain of RePhaC1 (RePhaC1ND ) is located opposite the dimerization subdomain of RePhaC1CD , indicating that RePhaC1ND is not directly involved in the enzyme catalysis. The localization studies using RePhaC1F , RePhaC1ND and RePhaC1CD revealed that RePhaC1ND plays important roles in PHA polymerization by localizing the enzyme to the PHA granules and stabilizing the growing PHA polymer near the active site of RePhaC1CD . The serial truncation study on RePhaC1ND suggested that the predicted five α-helices (N-α3 to N-α7) are required for proper folding and granule binding function of RePhaC1ND . In addition, the authors also report the SAXS 3D reconstructed model of the RePhaC1F /RePhaMΔC complex (RePhaMΔC , PAKKA motif-truncated version of RePhaM). RePhaM forms a complex with RePhaC1 by interacting with RePhaC1ND and activates RePhaC1 by providing a more extensive surface area for interaction with the growing PHA polymer.

  11. Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model.

    PubMed

    Rehm, Bernd H A; Antonio, Regina V; Spiekermann, Patricia; Amara, Amro A; Steinbüchel, Alexander

    2002-01-31

    A threading model of the Ralstonia eutropha polyhydroxyalkanoate (PHA) synthase was developed based on the homology to the Burkholderia glumae lipase, whose structure has been resolved by X-ray analysis. The lid-like structure in the model was discussed. In this study, various R. eutropha PHA synthase mutants were generated employing random as well as site-specific mutagenesis. Four permissive mutants (double and triple mutations) were obtained from single gene shuffling, which showed reduced activity and whose mutation sites mapped at variable surface-exposed positions. Six site-specific mutations were generated in order to identify amino acid residues which might be involved in substrate specificity. Replacement of residues T323 (I/S) and C438 (G), respectively, which are located in the core structure of the PHA synthase model, abolished PHA synthase activity. Replacement of the two amino acid residues Y445 (F) and L446 (K), respectively, which are located at the surface of the protein model and adjacent to W425, resulted in reduced activity without changing substrate specificity and indicating a functional role of these residues. The E267K mutant exhibited only slightly reduced activity with a surface-exposed mutation site. Four site-specific deletions were generated to evaluate the role of the C-terminus and variant amino acid sequence regions, which link highly conserved regions. Deleted regions were D281-D290, A372-C382, E578-A589 and V585-A589 and the respective PHA synthases showed no detectable activity, indicating an essential role of the variable C-terminus and the linking regions between conserved blocks 2 and 3 as well as 3 and 4. Moreover, the N-terminal part of the class II PHA synthase (PhaC(Pa)) from Pseudomonas aeruginosa and the C-terminal part of the class I PHA synthase (PhaC(Re)) from R. eutropha were fused, respectively, resulting in three fusion proteins with no detectable in vivo activity. However, the fusion protein F1 (PhaC(Pa)-1-265-Pha

  12. Evaluation of edible ginger and turmeric cultivars for root-knot nematode resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Edible ginger and turmeric roots are important agricultural commodities for the State of Hawaii. Bacterial wilt, Ralstonia solanacearum, and root-knot nematodes, Meloidogyne spp. are major factors hindering optimum production. An evaluation of tolerance and resistance to M. incognita was undertake...

  13. Antagonistic activity of Bacillus subtilis SB1 and its biocontrol effect on tomato bacterial wilt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, isolated from tomato roots, showed a broad-spectrum of antimicrobial activity in in vitro experiments. It inhibited the growth of many plant pathogens, including Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, Fusarium ox...

  14. Elevated temperature strain gages

    NASA Technical Reports Server (NTRS)

    Brittain, J. O.; Geslin, D.; Lei, J. F.

    1985-01-01

    Materials were evaluated that could be used in manufacturing electrical resistance strain gages for static strain measurements at temperatures at or above 1273 K. Strain gage materials must have a characteristic response to strain, temperature and time that is reproducible or that varies in a predictable manner within specified limits. Several metallic alloys were evaluated, as well as a series of transition metal carbides, nitrides and silicides.

  15. Sprains and Strains

    MedlinePlus

    ... happens. A strain is a stretched or torn muscle or tendon. Tendons are tissues that connect muscle to bone. Twisting or pulling these tissues can ... suddenly or develop over time. Back and hamstring muscle strains are common. Many people get strains playing ...

  16. To be or not to be a poly(3-hydroxybutyrate) (PHB) depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, highly active PHB depolymerases with no detectable role in mobilization of accumulated PHB.

    PubMed

    Sznajder, Anna; Jendrossek, Dieter

    2014-08-01

    The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure.

  17. Strain powered antennas

    NASA Astrophysics Data System (ADS)

    Domann, John P.; Carman, Greg P.

    2017-01-01

    This paper proposes the creation of strain powered antennas that radiate electromagnetic energy by mechanically vibrating a piezoelectric or piezomagnetic material. A closed form analytic model of electromagnetic radiation from a strain powered electrically small antenna is derived and analyzed. Fundamental scaling laws and the frequency dependence of strain powered antennas are discussed. The radiation efficiency of strain powered electrically small antennas is contrasted with a conventional electric dipole. Analytical results show that operating at the first mechanical resonance produces the most efficient strain powered radiation relative to electric dipole antennas. A resonant analysis is exploited to determine the material property space that produces efficient strain powered antennas. These results show how a properly designed strain powered antenna can radiate more efficiently than an equally sized electric dipole antenna.

  18. Expression of the Nitroarene Dioxygenase Genes in Comamonas sp. Strain JS765 and Acidovorax sp. Strain JS42 Is Induced by Multiple Aromatic Compounds

    PubMed Central

    Lessner, Daniel J.; Parales, Rebecca E.; Narayan, Shakti; Gibson, David T.

    2003-01-01

    This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator. PMID:12813084

  19. Pathway and Evolutionary Implications of Diphenylamine Biodegradation by Burkholderia sp. Strain JS667▿

    PubMed Central

    Shin, Kwanghee A.; Spain, Jim C.

    2009-01-01

    Diphenylamine (DPA) is a common contaminant at munitions-contaminated sites as well as at aniline manufacturing sites. Little is known about the biodegradation of the compound, and bacteria able to use DPA as the growth substrate have not been reported. Burkholderia sp. strain JS667 and Ralstonia sp. strain JS668 were isolated by selective enrichment from DPA-contaminated sediment. The isolates grew aerobically with DPA as the sole carbon, nitrogen, and energy source. During induction of DPA degradation, stoichiometric amounts of aniline accumulated and then disappeared, which suggested that aniline is on the DPA degradation pathway. Genes encoding the enzymes that catalyze the initial steps in DPA degradation were cloned from the genomic DNA of strain JS667. The Escherichia coli clone catalyzed stoichiometric transformation of DPA to aniline and catechol. Transposon mutagenesis, the sequence similarity of putative open reading frames to those of well-characterized dioxygenases, and 18O2 experiments support the conclusion that the initial reaction in DPA degradation is catalyzed by a multicomponent ring-hydroxylating dioxygenase. DPA is converted to aniline and catechol via dioxygenation at the 1,2 position of the aromatic ring and spontaneous rearomatization. Aniline and catechol are further biodegraded by the well-established aniline degradation pathway. Genes that encode the complete aniline degradation pathway were found 12 kb downstream of the genes that encode the initial dioxygenase. Expression of the relevant dioxygenases was confirmed by reverse transcription-PCR analysis. Both the sequence similarity and the gene organization suggest that the DPA degradation pathway evolved recently by the recruitment of two gene clusters that encode the DPA dioxygenase and aniline degradation pathway. PMID:19251893

  20. Three dimensional strained semiconductors

    DOEpatents

    Voss, Lars; Conway, Adam; Nikolic, Rebecca J.; Leao, Cedric Rocha; Shao, Qinghui

    2016-11-08

    In one embodiment, an apparatus includes a three dimensional structure comprising a semiconductor material, and at least one thin film in contact with at least one exterior surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the three dimensional structure. In another embodiment, a method includes forming a three dimensional structure comprising a semiconductor material, and depositing at least one thin film on at least one surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the structure.

  1. Strain sensor comprising a strain sensitive, two-mode optical

    NASA Technical Reports Server (NTRS)

    Egalon, Claudio Oliveira (Inventor); Rogowski, Robert S. (Inventor)

    1994-01-01

    A strain sensor uses an optical fiber including a strain sensitive portion and at least one strain insensitive portion. The strain sensitive portion is mounted on the surface of a structure at a location where a strain is desired to be measured. The strain insensitive portion(s) may be fused to the strain sensitive portion to transmit light therethrough, so that the resulting pattern may be detected to determine the amount of strain by comparison with a similar fiber not subjected to strain, or with the light pattern produced when the fiber is not under strain.

  2. Thin film strain transducer

    NASA Technical Reports Server (NTRS)

    Rand, J. L. (Inventor)

    1984-01-01

    A strain transducer system and process for making the same is disclosed. A beryllium copper ring having four strain gages is electrically connected in Wheatstone bridge fashion to the output instrumentation. Tabs are bonded to a balloon or like surface with strain on the surface causing bending of a ring which provides an electrical signal through the gages proportional to the surface strain. A photographic pattern of a one half ring segment as placed on a sheet of beryllium copper for chem-mill etch formation is illustrated.

  3. Mechanical strain isolator mount

    NASA Technical Reports Server (NTRS)

    James, Gordon E. (Inventor)

    1991-01-01

    Certain devices such as optical instruments must preserve their alignmental integrity while being subjected to mechanical strain. A mechanical strain isolator mount is provided to preserve the alignmental integrity of an alignment sensitive instrument. An alignment sensitive instrument is mounted on a rectangular base. Flexural legs are connected at their proximal ends to the rectangular base. Flexural legs are also spaced parallel to the sides. Mounting pads are connected to the legs at the distal end and the mechanical strain isolator mount is attached to the substrate by means of threaded bolts. When a mounting pad and its respective leg is subjected to lateral strain in either the X or Y direction via the substrate, the respective leg relieves the strain by bending in the direction of the strain. An axial strain on a mounting pad in the Z direction is relieved by a rotational motion of the legs in the direction of the strain. When the substrate is stress free, the flexural legs return to their original condition and thus preserve the original alignment integrity of the alignment sensitive instrument.

  4. Light intensity strain analysis

    NASA Technical Reports Server (NTRS)

    Williams, J. G. (Inventor)

    1973-01-01

    A process is described for the analysis of the strain field of structures subjected to large deformations involving a low modulus substrate having a high modulus, relatively thin coating. The optical properties of transmittance and reflectance are measured for the coated substrate while stressed and unstressed to indicate the strain field for the coated substrate.

  5. Thin film strain transducer

    NASA Astrophysics Data System (ADS)

    Rand, J. L.

    1981-01-01

    Previous attempts to develop an appropriate sensor for measuring the stress or strain of high altitude balloons during flight are reviewed as well as the various conditions that must be met by such a device. The design, development and calibration of a transducer which promises to satisfy the necessary design constraints are described. The thin film strain transducer has a low effective modulus so as not to interfere with the strain that would naturally occur in the balloon. In addition, the transducer has a high sensitivity to longitudinal strain (7.216 mV/V/unit strain) which is constant for all temperature from room temperature to -80 C and all strains from 5 percent compression to 10 percent tensile strain. At the same time, the sensor is relatively insensitive (0.27 percent) to transverse forces. The device has a standard 350 ohm impedance which is compatible with available bridge balance, amplification and telemetry instrumentation now available for balloon flight. Recommendations are included for improved coatings to provide passive thermal control as well as model, tethered and full scale flight testing.

  6. Strain gauge installation tool

    DOEpatents

    Conard, Lisa Marie

    1998-01-01

    A tool and a method for attaching a strain gauge to a test specimen by maaining alignment of, and applying pressure to, the strain gauge during the bonding of the gauge to the specimen. The tool comprises rigid and compliant pads attached to a spring-loaded clamp. The pads are shaped to conform to the specimen surface to which the gauge is to be bonded. The shape of the pads permits the tool to align itself to the specimen and to maintain alignment of the gauge to the specimen during the bond curing process. A simplified method of attaching a strain gauge is provided by use of the tool.

  7. Sprains and Strains

    MedlinePlus

    ... Typically, people with a strain experience pain, limited motion, muscle spasms, and possibly muscle weakness. They also ... program designed to prevent stiffness, improve range of motion, and restore the joint's normal flexibility and strength. ...

  8. Strains and Sprains

    MedlinePlus

    ... in the joint or muscle swelling and bruising warmth and redness of the injured area difficulty moving ... looks "bent" or misshapen signs of infection (increased warmth, redness, streaks, swelling, and pain) a strain or ...

  9. Strain and magnetic remanence

    NASA Astrophysics Data System (ADS)

    Borradaile, Graham John

    1993-05-01

    Experimental data may be compatible with the hypothesis that a single direction of magnetic remanence rotates as a rigid marker with strains up to 40% shortening in coaxial, perfect flattening ( X = Y > Z). Detailed agreement with the passive line model is relatively poor for the specimens in which remanance is carried by magnetite. However, for this range of strains the differences with the passive line model (Wettstein's equation) are so slight that the latter model may be more easily employed to de-strain or restore deformed remanance to its original attitude. In the case of hematite-bearing remanences, the differences between the passive line and rigid marker model are even smaller because of the higher aspect ratios of grains of hematite. Therefore it is suggested that Wettstein's equation may be safely used to restore remanence after even higher strains, where the remanence is carried by hematite.

  10. A transposable class I composite transposon carrying mph (methyl parathion hydrolase) from Pseudomonas sp. strain WBC-3.

    PubMed

    Wei, Min; Zhang, Jun-Jie; Liu, Hong; Wang, Shu-Jun; Fu, He; Zhou, Ning-Yi

    2009-03-01

    Pseudomonas sp. strain WBC-3 utilizes methyl parathion (O,O-dimethyl O-p-nitrophenol phosphorothioate) or para-nitrophenol as the sole source of carbon, nitrogen and energy. A gene encoding methyl parathion hydrolase (MPH) had been characterized previously and found to be located on a typical class I composite transposon that comprised IS6100 (Tnmph). In this study, the transposability of this transposon was confirmed by transposition assays in two distinct mating-out systems. Tnmph was demonstrated to transpose efficiently in a random manner in Pseudomonas putida PaW340 by Southern blot and in Ralstonia sp. U2 by sequence analysis of the Tnmph insertion sites, both exhibiting MPH activity. The linkage of the mph-like gene with IS6100, together with the transposability of Tnmph, as well as its capability to transpose in other phylogenetically divergent bacterial species, suggest that Tnmph may contribute to the wide distribution of mph-like genes and the adaptation of bacteria to organophosphorus compounds.

  11. Genome-wide sequencing data reveals virulence factors implicated in banana Xanthomonas wilt.

    PubMed

    Studholme, David J; Kemen, Eric; MacLean, Daniel; Schornack, Sebastian; Aritua, Valente; Thwaites, Richard; Grant, Murray; Smith, Julian; Jones, Jonathan D G

    2010-09-01

    Banana Xanthomonas wilt is a newly emerging disease that is currently threatening the livelihoods of millions of farmers in East Africa. The causative agent is Xanthomonas campestris pathovar musacearum (Xcm), but previous work suggests that this pathogen is much more closely related to species Xanthomonas vasicola than to X. campestris. We have generated draft genome sequences for a banana-pathogenic strain of Xcm isolated in Uganda and for a very closely related strain of X. vasicola pathovar vasculorum, originally isolated from sugarcane, that is nonpathogenic on banana. The draft sequences revealed overlapping but distinct repertoires of candidate virulence effectors in the two strains. Both strains encode homologues of the Pseudomonas syringae effectors HopW, HopAF1 and RipT from Ralstonia solanacearum. The banana-pathogenic and non-banana-pathogenic strains also differed with respect to lipopolysaccharide synthesis and type-IV pili, and in at least several thousand single-nucleotide polymorphisms in the core conserved genome. We found evidence of horizontal transfer between X. vasicola and very distantly related bacteria, including members of other divisions of the Proteobacteria. The availability of these draft genomes will be an invaluable tool for further studies aimed at understanding and combating this important disease.

  12. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation

    PubMed Central

    Liu, Hongxia; Kolter, Roberto; Losick, Richard; Guo, Jian-hua

    2014-01-01

    Summary Bacillus subtilis and other Bacilli have long been used as biological control agents against plant bacterial diseases but the mechanisms by which the bacteria confer protection are not well understood. Our goal in this study was to isolate strains of B. subtilis that exhibit high levels of biocontrol efficacy from natural environments and to investigate the mechanisms by which these strains confer plant protection. We screened a total of sixty isolates collected from various locations across China and obtained six strains that exhibited above 50% biocontrol efficacy on tomato plants against the plant pathogen Ralstonia solanacearum under greenhouse conditions. These wild strains were able to form robust biofilms both in defined medium and on tomato plant roots and exhibited strong antagonistic activities against various plant pathogens in plate assays. We show that plant protection by those strains depended on widely conserved genes required for biofilm formation, including regulatory genes and genes for matrix production. We provide evidence suggesting that matrix production is critical for bacterial colonization on plant root surfaces. Finally, we have established a model system for studies of B. subtilis-tomato plant interactions in protection against a plant pathogen. PMID:22934631

  13. Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

    PubMed

    Trefault, N; De la Iglesia, R; Molina, A M; Manzano, M; Ledger, T; Pérez-Pantoja, D; Sánchez, M A; Stuardo, M; González, B

    2004-07-01

    Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.

  14. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Bauchan, Gary R; Murphy, Charles; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2015-01-01

    Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.

  15. Strain isolated ceramic coatings

    NASA Technical Reports Server (NTRS)

    Tolokan, R. P.; Brady, J. B.; Jarrabet, G. P.

    1985-01-01

    Plasma sprayed ceramic coatings are used in gas turbine engines to improve component temperature capability and cooling air efficiency. A compliant metal fiber strain isolator between a plasma sprayed ceramic coating and a metal substrate improves ceramic durability while allowing thicker coatings for better insulation. Development of strain isolated coatings has concentrated on design and fabrication of coatings and coating evaluation via thermal shock testing. In thermal shock testing, five types of failure are possible: buckling failure im compression on heat up, bimetal type failure, isothermal expansion mismatch failure, mudflat cracking during cool down, and long term fatigue. A primary failure mode for thermally cycled coatings is designated bimetal type failure. Bimetal failure is tensile failure in the ceramic near the ceramic-metal interface. One of the significant benefits of the strain isolator is an insulating layer protecting the metal substrate from heat deformation and thereby preventing bimetal type failure.

  16. Strain avalanches in plasticity

    NASA Astrophysics Data System (ADS)

    Argon, A. S.

    2013-09-01

    Plastic deformation at the mechanism level in all solids occurs in the form of discrete thermally activated individual stress relaxation events. While there are clear differences in mechanisms between dislocation mediated events in crystalline solids and by individual shear transformations in amorphous metals and semiconductors, such relaxation events interact strongly to form avalanches of strain bursts. In all cases the attendant distributions of released energy as amplitudes of acoustic emissions, or in serration amplitudes in flow stress, the levels of strain bursts are of fractal character with fractal exponents in the range from -1.5 to -2.0, having the character of phenomena of self-organized criticality, SOC. Here we examine strain avalanches in single crystals of ice, hcp metals, the jerky plastic deformations of nano-pillars of fcc and bcc metals deforming in compression, those in the plastic flow of bulk metallic glasses, all demonstrating the remarkable universality of character of plastic relaxation events.

  17. Strain gauge installation tool

    DOEpatents

    Conard, L.M.

    1998-06-16

    A tool and a method are disclosed for attaching a strain gauge to a test specimen by maintaining alignment of, and applying pressure to, the strain gauge during the bonding of the gauge to the specimen. The tool comprises rigid and compliant pads attached to a spring-loaded clamp. The pads are shaped to conform to the specimen surface to which the gauge is to be bonded. The shape of the pads permits the tool to align itself to the specimen and to maintain alignment of the gauge to the specimen during the bond curing process. A simplified method of attaching a strain gauge is provided by use of the tool. 6 figs.

  18. Radio frequency strain monitor

    NASA Technical Reports Server (NTRS)

    Heyman, Joseph S. (Inventor); Rogowski, Robert S. (Inventor); Holben, Jr., Milford S. (Inventor)

    1989-01-01

    A radio frequency strain monitor includes a voltage controlled oscillator for generating an oscillating signal that is input into a propagation path. The propagation path is preferably bonded to the surface of a structure to be monitored and produces a propagated signal. A phase difference between the oscillating and propagated signals is detected and maintained at a substantially constant value which is preferably a multiple of 90.degree. by changing the frequency of the oscillating signal. Any change in frequency of the oscillating signal provides an indication of strain in the structure to which the propagation path is bonded.

  19. Muscle strain injuries.

    PubMed

    Garrett, W E

    1996-01-01

    One of the most common injuries seen in the office of the practicing physician is the muscle strain. Until recently, little data were available on the basic science and clinical application of this basic science for the treatment and prevention of muscle strains. Studies in the last 10 years represent action taken on the direction of investigation into muscle strain injuries from the laboratory and clinical fronts. Findings from the laboratory indicate that certain muscles are susceptible to strain injury (muscles that cross multiple joints or have complex architecture). These muscles have a strain threshold for both passive and active injury. Strain injury is not the result of muscle contraction alone, rather, strains are the result of excessive stretch or stretch while the muscle is being activated. When the muscle tears, the damage is localized very near the muscle-tendon junction. After injury, the muscle is weaker and at risk for further injury. The force output of the muscle returns over the following days as the muscle undertakes a predictable progression toward tissue healing. Current imaging studies have been used clinically to document the site of injury to the muscle-tendon junction. The commonly injured muscles have been described and include the hamstring, the rectus femoris, gastrocnemius, and adductor longus muscles. Injuries inconsistent with involvement of a single muscle-tendon junction proved to be at tendinous origins rather than within the muscle belly. Important information has also been provided regarding injuries with poor prognosis, which are potentially repairable surgically, including injuries to the rectus femoris muscle, the hamstring origin, and the abdominal wall. Data important to the management of common muscle injuries have been published. The risks of reinjury have been documented. The early efficacy and potential for long-term risks of nonsteroidal antiinflammatory agents have been shown. New data can also be applied to the field

  20. Sadovskii vortex in strain

    NASA Astrophysics Data System (ADS)

    Freilich, Daniel; Llewellyn Smith, Stefan

    2015-11-01

    Sadovskii vortices are patches of fluid with uniform vorticity surrounded by a vortex sheet. They were first constructed as models for wakes behind bluff objects. We investigate the Sadovskii vortex in a straining field and examine limiting cases to validate our computational method. One limit is the patch vortex in strain (Moore & Saffman, Aircraft wake turbulence and its detection 1971), where there is no vortex sheet. We solve this as a free-boundary problem, and show that a simple method using the Biot-Savart law quickly gives solutions for stable shapes. When used for the more elongated (stronger straining field) situations, the method also leads to new vortex shapes. In the hollow vortex case, where there is no vortex patch and the circulation is entirely due to the vortex sheet (Llewellyn Smith and Crowdy, J. Fluid Mech. 691 2012), we use the Birkhoff-Rott equation to calculate the velocity of the fluid on the vortex boundary. The combination of these two methods can then be used to calculate the shape and velocity field of the Sadovksii vortex in strain.

  1. Strain gage barometric transmitter

    NASA Technical Reports Server (NTRS)

    Viton, P.

    1977-01-01

    A strain gage barometric transmitter for measuring the atmospheric pressure in severe environmental conditions is described. This equipment specifications are presented and its performance assessed. It is shown that this barometric sensor can measure the atmospheric pressure with a precision of 0.5 mb during a 6 month period.

  2. Hamstring strain - aftercare

    MedlinePlus

    ... not to push yourself too hard or too fast. A hamstring strain can recur, or your hamstring may tear. Talk to your provider before returning to work or any physical activity. Returning to normal activity too early can cause re-injury.

  3. Repetitive strain injury.

    PubMed

    Al-Otaibi, S T

    2001-05-01

    Repetitive strain injury is a group of musculoskeletal disorders affecting muscles, tendons, nerves and blood vessels. These disorders could be attributed to occupational causes; however non-occupational causes should be excluded. The management of these cases required a multidisciplinary team approach.

  4. Balloon film strain measurement

    NASA Astrophysics Data System (ADS)

    Rand, James L.

    In order to understand the state of stress in scientific balloons, a need exists for the measurement of film deformation in flight. The results of a flight test program are reported where material strain was measured for the first time during the inflation, launch, ascent and float of a typical natural shape, zero pressure scientific balloon.

  5. Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophages: Two possible strategies for improving bacteriophage persistence for plant disease control.

    PubMed

    Iriarte, Fanny B; Obradović, Aleksa; Wernsing, Mine H; Jackson, Lee E; Balogh, Botond; Hong, Jason A; Momol, M Timur; Jones, Jeffrey B; Vallad, Gary E

    2012-10-01

    Soil-based root applications and attenuated bacterial strains were evaluated as means to enhance bacteriophage persistence on plants for bacterial disease control. In addition, the systemic nature of phage applied to tomato roots was also evaluated. Several experiments were conducted applying either single phages or phage mixtures specific for Ralstonia solanacearum, Xanthomonas perforans or X. euvesicatoria to soil surrounding tomato plants and measuring the persistence and translocation of the phages over time. In general, all phages persisted in the roots of treated plants and were detected in stems and leaves; although phage level varied and persistence in stems and leaves was at a much lower level compared with persistence in roots. Bacterial wilt control was typically best if the phage or phage mixtures were applied to the soil surrounding tomatoes at the time of inoculation, less effective if applied 3 days before inoculation, and ineffective if applied 3 days after inoculation. The use of an attenuated X. perforans strain was also evaluated to improve the persistence of phage populations on tomato leaf surfaces. In greenhouse and field experiments, foliar applications of an attenuated mutant X. perforans 91-118:∆OPGH strain prior to phage applications significantly improved phage persistence on tomato foliage compared with untreated tomato foliage. Both the soil-based bacteriophage delivery and the use of attenuated bacterial strains improved bacteriophage persistence on respective root and foliar tissues, with evidence of translocation with soil-based bacteriophage applications. Both strategies could lead to improved control of bacterial pathogens on plants.

  6. Highly stretchable miniature strain sensor for large dynamic strain measurement

    DOE PAGES

    Song, Bo; Yao, Shurong; Nie, Xu; ...

    2016-01-01

    In this paper, a new type of highly stretchable strain sensor was developed to measure large strains. The sensor was based on the piezo-resistive response of carbon nanotube (CNT)/polydimethylsiloxane (PDMS) composite thin films. The piezo-resistive response of CNT composite gives accurate strain measurement with high frequency response, while the ultra-soft PDMS matrix provides high flexibility and ductility for large strain measurement. Experimental results show that the CNT/PDMS sensor measures large strains (up to 8 %) with an excellent linearity and a fast frequency response. The new miniature strain sensor also exhibits much higher sensitivities than the conventional foil strain gages,more » as its gauge factor is 500 times of that of the conventional foil strain gages.« less

  7. Highly stretchable miniature strain sensor for large dynamic strain measurement

    SciTech Connect

    Song, Bo; Yao, Shurong; Nie, Xu; Yu, Xun; Blecke, Jill

    2016-01-01

    In this paper, a new type of highly stretchable strain sensor was developed to measure large strains. The sensor was based on the piezo-resistive response of carbon nanotube (CNT)/polydimethylsiloxane (PDMS) composite thin films. The piezo-resistive response of CNT composite gives accurate strain measurement with high frequency response, while the ultra-soft PDMS matrix provides high flexibility and ductility for large strain measurement. Experimental results show that the CNT/PDMS sensor measures large strains (up to 8 %) with an excellent linearity and a fast frequency response. The new miniature strain sensor also exhibits much higher sensitivities than the conventional foil strain gages, as its gauge factor is 500 times of that of the conventional foil strain gages.

  8. Strain patterns and strain accumulation along plate margins

    NASA Technical Reports Server (NTRS)

    Savage, J. C.

    1978-01-01

    Observations of strain accumulation along plate margins in Japan, New Zealand, and the United States indicate that: (1) a typical maximum rate of secular strain accumulation is on the order of 0.3 ppm/a, (2) a substantial part of the strain accumulation process can be attributed to slip at depth on the major plate boundary faults, and (3) some plastic deformation in a zone 100 km or more in width is apparently involved in the strain accumulation process.

  9. High temperature strain gages

    NASA Technical Reports Server (NTRS)

    Gregory, Otto J. (Inventor); You, Tao (Inventor)

    2011-01-01

    A ceramic strain gage based on reactively sputtered indium-tin-oxide (ITO) thin films is used to monitor the structural integrity of components employed in aerospace propulsion systems operating at temperatures in excess of 1500.degree. C. A scanning electron microscopy (SEM) of the thick ITO sensors reveals a partially sintered microstructure comprising a contiguous network of submicron ITO particles with well defined necks and isolated nanoporosity. Densification of the ITO particles was retarded during high temperature exposure with nitrogen thus stabilizing the nanoporosity. ITO strain sensors were prepared by reactive sputtering in various nitrogen/oxygen/argon partial pressures to incorporate more nitrogen into the films. Under these conditions, sintering and densification of the ITO particles containing these nitrogen rich grain boundaries was retarded and a contiguous network of nano-sized ITO particles was established.

  10. Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly (3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses

    PubMed Central

    2013-01-01

    Background Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16. Results Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for β-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition. Conclusions The results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha. PMID:23879744

  11. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

    PubMed

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

    A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.

  12. The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound [NiFe] Hydrogenase in Ralstonia eutropha H16 ▿ †

    PubMed Central

    Fritsch, Johannes; Lenz, Oliver; Friedrich, Bärbel

    2011-01-01

    The membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O2 and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O2 levels. MBH-driven growth on H2 is inhibited or retarded at high O2 partial pressure (pO2) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO2. Lack of hoxR and hoxT can phenotypically be restored by providing O2-limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions. PMID:21441514

  13. Strained Ring Energetic Binders

    DTIC Science & Technology

    1993-08-27

    polyhomobenzvalene ( PHBV ). PHBV was not found to have the mechanical instability problems of PBV, but was still thermally unstable (Tonset - 660C, Tmax - 1090C...DISCUSSION 4 Polybenzvalene (PBV) 4 Polyhomobenzvalene ( PHBV ) 6 Chain-Transfer Studies 11 CONCLUSIONS 15 EXPERIMENTAL PROCEDURES 16 .F 4E 19 APPENDICES A...strained ring polymers similar to PBV are known. The investigation of one of these polymers, polyhomobenzvalene ( PHBV ), is also described in this report

  14. Strain Gage Signal Interpretation.

    DTIC Science & Technology

    1986-02-01

    blades and vanes in many engines have been collected, played back and examined. The engine types encompass GE’s stable of turbine engines from the small...aeromechanical engineer . 1.3 SUMMARY OF RESULTS Strain gage signals from vibrating rotor blades and vanes were collected, examined, classified, and generalized...turboprops, to turbojets and to the large high bypass turbofan engines . Test conditions include all the phases that are investigated

  15. Strain Measurement - Unidirectional.

    DTIC Science & Technology

    1983-04-20

    a ballpoint pen or a rounded piece of brass rod. If critical alignment is not necessary, gage lines may be located outside the immediate gage location...supplemented as needed by tabulated values. If the test design includes specification limits for the values, they should be included on the plots. Plots of...enough baseline before the event to allow estimation of the noise and stability. If the strain is to be correlated to specific events, the events should

  16. What Are Sprains and Strains?

    MedlinePlus

    ... hands and arms a lot. Examples are gymnastics, tennis, rowing, and golf. People who play these sports sometimes strain their hand or arm. Elbow strains can also happen when playing sports. What ...

  17. Priming by rhizobacterium protects tomato plants from biotrophic and necrotrophic pathogen infections through multiple defense mechanisms.

    PubMed

    Ahn, Il-Pyung; Lee, Sang-Woo; Kim, Min Gab; Park, Sang-Ryeol; Hwang, Duk-Ju; Bae, Shin-Chul

    2011-07-01

    A selected strain of rhizobacterium, Pseudomonas putida strain LSW17S (LSW17S), protects tomato plants (Lycopersicon esculentum L. cv. Seokwang) from bacterial speck by biotrophic Pseudomonas syringae pv. tomato strain DC3000 (DC3000) and bacterial wilt by necrotrophic Ralstonia solanacearum KACC 10703 (Rs10703). To investigate defense mechanisms induced by LSW17S in tomato plants, transcription patterns of pathogenesis-related (PR) genes and H(2)O(2) production were analyzed in plants treated with LSW17S and subsequent pathogen inoculation. LSW17S alone did not induce transcriptions of employed PR genes in leaves and roots. DC3000 challenge following LSW17S triggered rapid transcriptions of PR genes and H(2)O(2) production in leaves and roots. Catalase infiltration with DC3000 attenuated defense-related responses and resistance against DC3000 infection. Despite depriving H(2)O(2) production and PR1b transcription by the same treatment, resistance against Rs10703 infection was not deterred significantly. H(2)O(2) is indispensable for defense signaling and/or mechanisms primed by LSW17S and inhibition of bacterial speck, however, it is not involved in resistance against bacterial wilt.

  18. Strain balanced quantum posts

    SciTech Connect

    Alonso-Alvarez, D.; Alen, B.; Ripalda, J. M.; Llorens, J. M.; Taboada, A. G.; Briones, F.; Roldan, M. A.; Hernandez-Saz, J.; Hernandez-Maldonado, D.; Herrera, M.; Molina, S. I.

    2011-04-25

    Quantum posts are assembled by epitaxial growth of closely spaced quantum dot layers, modulating the composition of a semiconductor alloy, typically InGaAs. In contrast with most self-assembled nanostructures, the height of quantum posts can be controlled with nanometer precision, up to a maximum value limited by the accumulated stress due to the lattice mismatch. Here, we present a strain compensation technique based on the controlled incorporation of phosphorous, which substantially increases the maximum attainable quantum post height. The luminescence from the resulting nanostructures presents giant linear polarization anisotropy.

  19. Sadovskii vortex in strain

    NASA Astrophysics Data System (ADS)

    Freilich, Daniel; Llewellyn Smith, Stefan

    2014-11-01

    A Sadovskii vortex is a patch of fluid with uniform vorticity surrounded by a vortex sheet. Using a boundary element type method, we investigate the steady states of this flow in an incompressible, inviscid straining flow. Outside the vortex, the fluid is irrotational. In the limiting case where the entire circulation is due to the vortex patch, this is a patch vortex (Moore & Saffman, Aircraft wake turbulence and its detection 1971). In the other limiting case, where all the circulation is due to the vortex sheet, this is a hollow vortex (Llewellyn Smith and Crowdy, J. Fluid Mech. 691, 2012). This flow has two governing nondimensional parameters, relating the strengths of the straining field, vortex sheet, and patch vorticity. We study the relationship between these two parameters, and examine the shape of the resulting vortices. We also work towards a bifurcation diagram of the steady states of the Sadovskii vortex in an attempt to understand the connection between vortex sheet and vortex patch desingularizations of the point vortex. Support from NSF-CMMI-0970113.

  20. Repetitive strain injury.

    PubMed

    van Tulder, Maurits; Malmivaara, Antti; Koes, Bart

    2007-05-26

    Repetitive strain injury remains a controversial topic. The term repetitive strain injury includes specific disorders such as carpal tunnel syndrome, cubital tunnel syndrome, Guyon canal syndrome, lateral epicondylitis, and tendonitis of the wrist or hand. The diagnosis is usually made on the basis of history and clinical examination. Large high-quality studies using newer imaging techniques, such as MRI and ultrasonography are few. Consequently, the role of such imaging in diagnosis of upper limb disorders remains unclear. In many cases, no specific diagnosis can be established and complaints are labelled as non-specific. Little is known about the effectiveness of treatment options for upper limb disorders. Strong evidence for any intervention is scarce and the effect, if any, is mainly short-term pain relief. Exercise is beneficial for non-specific upper limb disorders. Immobilising hand braces and open carpal tunnel surgery release are beneficial for carpal tunnel syndrome, and topical and oral non-steroidal anti-inflammatory drugs, and corticosteroid injections are helpful for lateral epicondylitis. Exercise is probably beneficial for neck pain, as are corticosteroid injections and exercise for shoulder pain. Although upper limb disorders occur frequently in the working population, most trials have not exclusively included a working population or assessed effects on work-related outcomes. Further high-quality trials should aim to include sufficient sample sizes, working populations, and work-related outcomes.

  1. Strain Pattern in Supercooled Liquids

    NASA Astrophysics Data System (ADS)

    Illing, Bernd; Fritschi, Sebastian; Hajnal, David; Klix, Christian; Keim, Peter; Fuchs, Matthias

    2016-11-01

    Investigations of strain correlations at the glass transition reveal unexpected phenomena. The shear strain fluctuations show an Eshelby-strain pattern [˜cos (4 θ ) /r2 ], characteristic of elastic response, even in liquids, at long times. We address this using a mode-coupling theory for the strain fluctuations in supercooled liquids and data from both video microscopy of a two-dimensional colloidal glass former and simulations of Brownian hard disks. We show that the long-ranged and long-lived strain signatures follow a scaling law valid close to the glass transition. For large enough viscosities, the Eshelby-strain pattern is visible even on time scales longer than the structural relaxation time τ and after the shear modulus has relaxed to zero.

  2. Hydrogen production from microbial strains

    DOEpatents

    Harwood, Caroline S; Rey, Federico E

    2012-09-18

    The present invention is directed to a method of screening microbe strains capable of generating hydrogen. This method involves inoculating one or more microbes in a sample containing cell culture medium to form an inoculated culture medium. The inoculated culture medium is then incubated under hydrogen producing conditions. Once incubating causes the inoculated culture medium to produce hydrogen, microbes in the culture medium are identified as candidate microbe strains capable of generating hydrogen. Methods of producing hydrogen using one or more of the microbial strains identified as well as the hydrogen producing strains themselves are also disclosed.

  3. Strain relaxation in nanopatterned strained silicon round pillars

    NASA Astrophysics Data System (ADS)

    Himcinschi, C.; Singh, R.; Radu, I.; Milenin, A. P.; Erfurth, W.; Reiche, M.; Gösele, U.; Christiansen, S. H.; Muster, F.; Petzold, M.

    2007-01-01

    Periodic arrays of strained Si (sSi) round nanopillars were fabricated on sSi layers deposited on SiGe virtual substrates by electron-beam lithography and subsequent reactive-ion etching. The strain in the patterned sSi nanopillars was determined using high-resolution UV micro-Raman spectroscopy. The strain relaxes significantly upon nanostructuring: from 0.9% in the unpatterned sSi layer to values between 0.22% and 0.57% in the round sSi pillars with diameters from 100 up to 500nm. The strain distribution in the sSi nanopillars was analyzed by finite element (FE) modeling. The FE calculations confirm the strain relaxation after patterning, in agreement with the results obtained from Raman spectroscopy.

  4. Adaptor for Measuring Principal Strains with Tuckerman Strain Gage

    NASA Technical Reports Server (NTRS)

    Mcpherson, A E

    1943-01-01

    An adapter is described which uses three Tuckerman optical strain gages to measure the displacement of the three vortices of an equilateral triangle along lines 120 degrees apart. These displacements are substituted in well-known equations in order to compute the magnitude and direction of the principal strains. Tests of the adaptor indicate that principal strains over a gage length of 1.42 inch may be measured with a systematic error not exceeding 4 percent and a mean observational error of the order of + or minus 0.000006. The maximum observed error in strain was of the order of 0.00006. The directions of principal strains for unidirectional stress were measured with the adaptor with an average error of the order of 1 degree.

  5. Production of bacillomycin- and macrolactin-type antibiotics by Bacillus amyloliquefaciens NJN-6 for suppressing soilborne plant pathogens.

    PubMed

    Yuan, Jun; Li, Bing; Zhang, Nan; Waseem, Raza; Shen, Qirong; Huang, Qiwei

    2012-03-28

    Bacillus amyloliquefaciens strains have been used as biocontrol agents for the suppression of several soilborne plant pathogens. A clearer understanding of the antagonistic mechanisms of action of these bacteria will facilitate their use in the control of plant diseases. Antagonistic substances were isolated from the fermentation broth of B. amyloliquefaciens strain NJN-6 cultures. These compounds were preconcentrated using an XAD-16 column and were purified using reversed-phase high-performance liquid chromatography (RP-HPLC). Fractions were collected from the column and were analyzed, and two homologues of bacillomycin D [molecular weights of 1030 Da (C14) and 1044 Da (C15)] and three homologues of members of the macrolactin family, macrolactin A, 7-O-malonyl macrolactin A, and 7-O-succinyl macrolactin A (molecular weights of 402, 487, and 502 Da, respectively) were identified using HPLC/electrospray ionization mass spectrometry (ESI-MS) analysis. An antagonistic assay showed that bacillomycin D and macrolactin exhibited significant antagonistic effects against Fusarium oxysporum and Ralstonia solanacearum , respectively. A reliable method for the isolation and purification of bacillomycin D and macrolactin from bacterial broth cultures was developed. These data will help elucidate the mechanisms that B. amyloliquefaciens NJN-6 uses for the biocontrol of soilborne plant pathogens.

  6. Identification and functional analysis of type III effector proteins in Mesorhizobium loti.

    PubMed

    Okazaki, Shin; Okabe, Saori; Higashi, Miku; Shimoda, Yoshikazu; Sato, Shusei; Tabata, Satoshi; Hashiguchi, Masatsugu; Akashi, Ryo; Göttfert, Michael; Saeki, Kazuhiko

    2010-02-01

    Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, possesses a cluster of genes (tts) that encode a type III secretion system (T3SS). In the presence of heterologous nodD from Rhizobium leguminosarum and a flavonoid naringenin, we observed elevated expression of the tts genes and secretion of several proteins into the culture medium. Inoculation experiments with wild-type and T3SS mutant strains revealed that the presence of the T3SS affected nodulation at a species level within the Lotus genus either positively (L. corniculatus subsp. frondosus and L. filicaulis) or negatively (L. halophilus and two other species). By inoculating L. halophilus with mutants of various type III effector candidate genes, we identified open reading frame mlr6361 as a major determinant of the nodulation restriction observed for L. halophilus. The predicted gene product of mlr6361 is a protein of 3,056 amino acids containing 15 repetitions of a sequence motif of 40 to 45 residues and a shikimate kinase-like domain at its carboxyl terminus. Homologues with similar repeat sequences are present in the hypersensitive-response and pathogenicity regions of several plant pathogens, including strains of Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas species. These results suggest that L. halophilus recognizes Mlr6361 as potentially pathogen derived and subsequently halts the infection process.

  7. Bacillus and Streptomyces were selected as broad-spectrum antagonists against soilborne pathogens from arid areas in Egypt.

    PubMed

    Köberl, Martina; Ramadan, Elshahat M; Adam, Mohamed; Cardinale, Massimiliano; Hallmann, Johannes; Heuer, Holger; Smalla, Kornelia; Berg, Gabriele

    2013-05-01

    Plant protection via disease-suppressive bacteria in desert farming requires specific biological control agents (BCAs) adapted to the unique arid conditions. We performed an ecological study of below-ground communities in desert farm soil and untreated desert soil, and based on these findings, selected antagonists were hierarchically evaluated. In contrast to the highly specific 16S rRNA fingerprints of bacterial communities in soil and cultivated medicinal plants, internal transcribed spacer profiles of fungal communities were less discriminative and mainly characterised by potential pathogens. Therefore, we focused on in vitro bacterial antagonists against pathogenic fungi. Based on the antifungal potential and genomic diversity, 45 unique strains were selected and characterised in detail. Bacillus/Paenibacillus were most frequently identified from agricultural soil, but antagonists from the surrounding desert soil mainly belonged to Streptomyces. All strains produced antibiotics against the nematode Meloidogyne incognita, and one-third showed additional activity against the bacterial pathogen Ralstonia solanacearum. Altogether, 13 broad-spectrum antagonists with antibacterial, antifungal and nematicidal activity were found. They belong to seven different bacterial species of the genera Bacillus and Streptomyces. These Gram-positive, spore-forming bacteria are promising drought-resistant BCAs and a potential source for antibiotics. Their rhizosphere competence was shown by fluorescence in situ hybridisation combined with laser scanning microscopy.

  8. Strained graphene Hall bar

    NASA Astrophysics Data System (ADS)

    Milovanović, S. P.; Peeters, F. M.

    2017-02-01

    The effects of strain, induced by a Gaussian bump, on the magnetic field dependent transport properties of a graphene Hall bar are investigated. The numerical simulations are performed using both classical and quantum mechanical transport theory and we found that both approaches exhibit similar characteristic features. The effects of the Gaussian bump are manifested by a decrease of the bend resistance, R B, around zero-magnetic field and the occurrence of side-peaks in R B. These features are explained as a consequence of bump-assisted scattering of electrons towards different terminals of the Hall bar. Using these features we are able to give an estimate of the size of the bump. Additional oscillations in R B are found in the quantum description that are due to the population/depopulation of Landau levels. The bump has a minor influence on the Hall resistance even for very high values of the pseudo-magnetic field. When the bump is placed outside the center of the Hall bar valley polarized electrons can be collected in the leads.

  9. Biomechanical strain of goldsmiths.

    PubMed

    Cândido, Paula Emanuela Fernandes; Teixeira, Juliana Vieira Schmidt; Moro, Antônio Renato Pereira; Gontijo, Leila Amaral

    2012-01-01

    The work of the goldsmiths consists in the manufacture of jewelry. The piece, be it an earring, bracelet or necklace, is hand-assembled. This task requires precision, skill, kindness and patience. In this work, we make use of tools such as cuticle clippers and rounded tip, beads or precious stones and also pieces of metal. This type of activity requires a biomechanical stress of hands and wrists. In order to quantify the biomechanical stress, we performed a case study to measure the movements performed by an assembly of pieces of jewelry. As method for research, filming was done during assembly of parts to a paste, using a Nikon digital camera, for 1 (one) hour. The film was edited by Kinovea software, and the task was divided into cycles, each cycle corresponds to a complete object. In one cycle, there are four two movements of supination and pronation movements of the forearm. The cycle lasts approximately sixteen seconds, totaling 1800 cycles in eight hours. Despite the effort required of the wrists, the activity shows no complaints from the employees, but this fact does not mischaracterizes the ability of employees to acquire repetitive strain injuries and work-related musculoskeletal disorders.

  10. Characterization of Salmonella enteritidis strains.

    PubMed Central

    Poppe, C; McFadden, K A; Brouwer, A M; Demczuk, W

    1993-01-01

    A study was conducted to characterize 318 Salmonella enteritidis strains that were mainly isolated from poultry and their environment in Canada. Biotype, phagetype (PT), plasmid profile (PP), hybridization with a plasmid-derived virulence sequence probe, antibiotic resistance, outer membrane proteins (OMPs), and lipopolysaccharide (LPS) profiles were determined. Relationships of these properties to one another, and their diagnostic and pathogenic significance were assessed. Biotyping indicated that failure to ferment rhamnose was sometimes useful as a marker for epidemiologically related strains. Phagetyping was the most effective method for subdividing S. enteritidis; it distinguished 12 PTs. Phagetype 13 was occasionally associated with septicemia and mortality in chickens. The strains belonged to 15 PPs. A 36 megadalton (MDa) plasmid was found in 97% of the strains. Only the 36 MDa plasmid hybridized with the probe. Seventeen percent of the strains were drug resistant; all strains were sensitive to ciprofloxacin. Thirty-five of 36 strains possessed the same OMP profile, and 36 of 41 strains contained smooth LPS. Images Fig. 1. PMID:8358678

  11. Difference Between Strain and Sprain.

    ERIC Educational Resources Information Center

    Connors, G. Patrick

    Provided in this description of the differences between a strain (damage to the muscle or tendon) and a sprain (damage to the ligament) are definitions of mild, moderate, and severe (first, second, and third degree) strains and sprains. A final caution is given that these are two separate and distinct problems and should be treated as such. (DC)

  12. Hypothetical strain-free oligoradicals

    PubMed Central

    Hoffmann, Roald; Eisenstein, Odile; Balaban, Alexandru T.

    1980-01-01

    Several new classes of oligoradicals free of angle strain are suggested and examined by means of molecular orbital calculations. The collapse products of these hypothetical radicals are highly strained molecules. Various electronic strategies for the stabilization of these oligoradicals have been explored. PMID:16592882

  13. Strain gage system evaluation program

    NASA Technical Reports Server (NTRS)

    Dolleris, G. W.; Mazur, H. J.; Kokoszka, E., Jr.

    1978-01-01

    A program was conducted to determine the reliability of various strain gage systems when applied to rotating compressor blades in an aircraft gas turbine engine. A survey of current technology strain gage systems was conducted to provide a basis for selecting candidate systems for evaluation. Testing and evaluation was conducted in an F 100 engine. Sixty strain gage systems of seven different designs were installed on the first and third stages of an F 100 engine fan. Nineteen strain gage failures occurred during 62 hours of engine operation, for a survival rate of 68 percent. Of the failures, 16 occurred at blade-to-disk leadwire jumps (84 percent), two at a leadwire splice (11 percent), and one at a gage splice (5 percent). Effects of erosion, temperature, G-loading, and stress levels are discussed. Results of a post-test analysis of the individual components of each strain gage system are presented.

  14. Strain Insensitive Optical Phase Locked Loop

    NASA Technical Reports Server (NTRS)

    Egalon, Claudio O. (Inventor); Rogowski, Robert S. (Inventor)

    1998-01-01

    A strain sensor uses optical fibers including strain insensitive portions and a strain sensitive portion. The optical fibers form a sensitive arm of an optical phase locked loop (OPLL). The use of the OPLL allows for multimode optical fiber to be used in a strain insensitive configuration. Only strain information for the strain sensitive portion is monitored rather than the integrated strain measurements commonly made with optical fiber sensors.

  15. High temperature strain measurement with a resistance strain gage

    NASA Technical Reports Server (NTRS)

    Lei, Jih-Fen; Fichtel, ED; Mcdaniel, Amos

    1993-01-01

    A PdCr based electrical resistance strain gage was demonstrated in the laboratory to be a viable sensor candidate for static strain measurement at high temperatures. However, difficulties were encountered while transferring the sensor to field applications. This paper is therefore prepared for recognition and resolution of the problems likely to be encountered with PdCr strain gages in field applications. Errors caused by the measurement system, installation technique and lead wire attachment are discussed. The limitations and some considerations related to the temperature compensation technique used for this gage are also addressed.

  16. Recent advances in echocardiography: strain and strain rate imaging

    PubMed Central

    Mirea, Oana; Duchenne, Jurgen; Voigt, Jens-Uwe

    2016-01-01

    Deformation imaging by echocardiography is a well-established research tool which has been gaining interest from clinical cardiologists since the introduction of speckle tracking. Post-processing of echo images to analyze deformation has become readily available at the fingertips of the user. New parameters such as global longitudinal strain have been shown to provide added diagnostic value, and ongoing efforts of the imaging societies and industry aimed at harmonizing methods will improve the technique further. This review focuses on recent advances in the field of echocardiographic strain and strain rate imaging, and provides an overview on its current and potential future clinical applications. PMID:27158476

  17. Low TCR nanocomposite strain gages

    NASA Technical Reports Server (NTRS)

    Gregory, Otto J. (Inventor); Chen, Ximing (Inventor)

    2012-01-01

    A high temperature thin film strain gage sensor capable of functioning at temperatures above 1400.degree. C. The sensor contains a substrate, a nanocomposite film comprised of an indium tin oxide alloy, zinc oxide doped with alumina or other oxide semiconductor and a refractory metal selected from the group consisting of Pt, Pd, Rh, Ni, W, Ir, NiCrAlY and NiCoCrAlY deposited onto the substrate to form an active strain element. The strain element being responsive to an applied force.

  18. Virtual strain gage size study

    SciTech Connect

    Reu, Phillip L.

    2015-09-22

    DIC is a non-linear low-pass spatial filtering operation; whether we consider the effect of the subset and shape function, the strain window used in the strain calculation, of other post-processing of the results, each decision will impact the spatial resolution, of the measurement. More fundamentally, the speckle size limits, the spatial resolution by dictating the smallest possible subset. After this decision the processing settings are controlled by the allowable noise level balanced by possible bias errors created by the data filtering. This article describes a process to determine optimum DIC software settings to determine if the peak displacements or strains are being found.

  19. Radio Frequency (RF) strain monitor

    NASA Technical Reports Server (NTRS)

    Heyman, Joseph S. (Inventor); Rogowski, Robert S. (Inventor); Holben, Milford S., Jr. (Inventor)

    1988-01-01

    This invention relates to an apparatus for measuring strain in a structure. In particular, the invention detects strain in parts per million to over ten percent along an entire length (or other dimension) of a structure measuring a few millimeters to several kilometers. By using a propagation path bonded to the structure, the invention is not limited by the signal attenuation characteristics of the structure and thus frequencies in the megahertz to gigahertz range may be used to detect strain in part per million to over ten percent with high precision.

  20. Strain accumulation in quasicrystalline solids

    NASA Technical Reports Server (NTRS)

    Nori, Franco; Ronchetti, Marco; Elser, Veit

    1988-01-01

    The relaxation of two-dimensional quasicrystalline elastic networks when their constituent bonds are perturbed homogeneously is studied. Whereas ideal, quasi-periodic networks are stable against such perturbations, significant accumulations of strain in a class of disordered networks generated by a growth process are found. The grown networks are characterized by root mean square phason fluctuations which grow linearly with system size. The strain accumulation observed in these networks also grows linearly with system size. Finally, dependence of strain accumulation on cooling rate is found.

  1. Production of rhamnolipids and diesel oil degradation by bacteria isolated from soil contaminated by petroleum.

    PubMed

    Leite, Giuseppe G F; Figueirôa, Juciane V; Almeida, Thiago C M; Valões, Jaqueline L; Marques, Walber F; Duarte, Maria D D C; Gorlach-Lira, Krystyna

    2016-03-01

    Biosurfactants are microbial secondary metabolites. The most studied are rhamnolipids, which decrease the surface tension and have emulsifying capacity. In this study, the production of biosurfactants, with emphasis on rhamnolipids, and diesel oil degradation by 18 strains of bacteria isolated from waste landfill soil contaminated by petroleum was analyzed. Among the studied bacteria, gram-positive endospore forming rods (39%), gram positive rods without endospores (17%), and gram-negative rods (44%) were found. The following methods were used to test for biosurfactant production: oil spreading, emulsification, and hemolytic activity. All strains showed the ability to disperse the diesel oil, while 77% and 44% of the strains showed hemolysis and emulsification of diesel oil, respectively. Rhamnolipids production was observed in four strains that were classified on the basis of the 16S rRNA sequences as Pseudomonas aeruginosa. Only those strains showed the rhlAB gene involved in rhamnolipids synthesis, and antibacterial activity against Escherichia coli, P. aeruginosa, Staphylococcus aureus, Bacillus cereus, Erwinia carotovora, and Ralstonia solanacearum. The highest production of rhamnolipids was 565.7 mg/L observed in mineral medium containing olive oil (pH 8). With regard to the capacity to degrade diesel oil, it was observed that 7 strains were positive in reduction of the dye 2,6-dichlorophenolindophenol (2,6-DCPIP) while 16 had the gene alkane mono-oxygenase (alkB), and the producers of rhamnolipids were positive in both tests. Several bacterial strains have shown high potential to be explored further for bioremediation purposes due to their simultaneous ability to emulsify, disperse, and degrade diesel oil. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:262-270, 2016.

  2. PLASTICITY AND NON-LINEAR ELASTIC STRAINS

    DTIC Science & Technology

    conditions existing in plane waves in an extended medium to give the time rate of change of stress as a function of the time rate of change of strain, the stress invariants, the total strain and the plastic strain. (Author)

  3. Magnetic Domain Strain Sensor Program

    DTIC Science & Technology

    1990-08-01

    static strain measurement at elevated temperatures. 2.2 Magnetic Strain Measurement Theory The initial work at GED investigated the Barkhausen effect...including large and small Barkhausen jumps. This is a wave propaga- tion phenomenon in which a magnetic wave velocity is measured. The wave velocity in a...theory explaining the phenomenon that deviates from the Barkhausen effect. Some basic concepts had to be examined to better understand magnetic phenomena

  4. HIGH-TEMPERATURE STRAIN GAGE,

    DTIC Science & Technology

    The patent involves a high-temperature tensometer consisting of a strain-sensitive wire grid, a connecting and insulating material, a sub-layer of...heat-resistant material, deposited on the part being investigated or on a backing by gas flame deposition, and a connector to fasten the strain...adhesion, the tension-sensitive wire grid is fastened through the sub-layer to the part being tested by the connecting and insulating material. (Author)

  5. NUTRITION OF FIVE BACTEROIDES STRAINS

    PubMed Central

    Quinto, Grace

    1962-01-01

    Quinto, Grace (University of Kentucky College of Medicine, Lexington). Nutrition of five Bacteroides strains. J. Bacteriol. 84:559–562. 1962.—Some of the nutritional requirements of five gram-negative anaerobic bacilli, including Ristella perfoetens, Zuberella clostridiformis, and three Bacteroides strains freshly isolated from clinical exudates, were investigated. A fluid maintenance medium was developed in which the three freshly isolated strains, C-4, C-7, and C-2795, grew maximally in 12 to 24 hr. The maintenance medium contained 2.0% Trypticase and Proteose Peptone, 0.5% glucose, and 0.1% sodium thioglycolate; it was adjusted to pH 7.2 and supplemented with 0.1 μg of hemin/ml. Strains C-4, C-7, and C-2795 were cultivated through 14 serial cultures in fluid maintenance medium containing 0.1 μg of hemin/ml. The most satisfactory inoculum was a 1:100 or 1:1,000 dilution of a 24-hr seed culture. All the strains except Z. clostridiformis grew serially in a defined medium. R. perfoetens required pantothenic acid, nicotinic acid, and the following amino acids: histidine, tryptophan, tyrosine, valine, phenylalanine, cystine, and probably arginine, glutamic acid, methionine, glycine, isoleucine, leucine, and lysine. The C-4, C-7, and C-2795 strains required the hemin supplement in defined medium, but not vitamins, purines, or pyrimidines. PMID:13972793

  6. Impact of a Bacterial Volatile 2,3-Butanediol on Bacillus subtilis Rhizosphere Robustness

    PubMed Central

    Yi, Hwe-Su; Ahn, Yeo-Rim; Song, Geun C.; Ghim, Sa-Youl; Lee, Soohyun; Lee, Gahyung; Ryu, Choong-Min

    2016-01-01

    Volatile compounds, such as short chain alcohols, acetoin, and 2,3-butanediol, produced by certain strains of root-associated bacteria (rhizobacteria) elicit induced systemic resistance in plants. The effects of bacterial volatile compounds (BVCs) on plant and fungal growth have been extensively studied; however, the impact of bacterial BVCs on bacterial growth remains poorly understood. In this study the effects of a well-characterized bacterial volatile, 2,3-butanediol, produced by the rhizobacterium Bacillus subtilis, were examined in the rhizosphere. The nature of 2,3-butanediol on bacterial cells was assessed, and the effect of the molecule on root colonization was also determined. Pepper roots were inoculated with three B. subtilis strains: the wild type, a 2,3-butanediol overexpressor, and a 2,3-butanediol null mutant. The B. subtilis null strain was the first to be eliminated in the rhizosphere, followed by the wild-type strain. The overexpressor mutant was maintained at roots for the duration of the experiment. Rhizosphere colonization by a saprophytic fungus declined from 14 days post-inoculation in roots treated with the B. subtilis overexpressor strain. Next, exudates from roots exposed to 2,3-butanediol were assessed for their impact on fungal and bacterial growth in vitro. Exudates from plant roots pre-treated with the 2,3-butanediol overexpressor were used to challenge various microorganisms. Growth was inhibited in a saprophytic fungus (Trichoderma sp.), the 2,3-butanediol null B. subtilis strain, and a soil-borne pathogen, Ralstonia solanacearum. Direct application of 2,3-butanediol to pepper roots, followed by exposure to R. solanacearum, induced expression of Pathogenesis-Related (PR) genes such as CaPR2, CaSAR8.2, and CaPAL. These results indicate that 2,3-butanediol triggers the secretion of root exudates that modulate soil fungi and rhizosphere bacteria. These data broaden our knowledge regarding bacterial volatiles in the rhizosphere and

  7. Applications of strained layer superlattices

    NASA Astrophysics Data System (ADS)

    Smith, D. L.; Laurich, B. K.; Mailhiot, C.

    1990-04-01

    Because of different band edge lineups, strain conditions, and growth orientations, various strained layer superlattice (SLS) materials can exhibit qualitatively new physical behavior in their optical properties. Two examples are given of new physical behavior in SLS: strain generated electric fields in polar growth axis superlattices and strained type 2 superlattices. In SLS, large electric fields can be generated by the piezoelectric effect. The fields are largest for SLS with a (111) growth axis; they vanish for SLS with a (100) growth axis. The strain generated electric fields strongly modify the optical properties of the superlattice. Photogenerated electron-hole pairs screen the fields leading to a large nonlinear optical response. Application of an external electric field leads to a large linear electrooptical response. The absorption edge can be either red or blue shifted. Optical studies of (100), (111), and (211) oriented GaInAs/GaAs superlattices confirm the existence of the strain generated electric fields. Small band gap semiconductors are useful for making intrinsic long wavelength infrared detectors. Arbitrarily small band gaps can be reached in the type 2 superlattice InAs/GaSb. However, for band gaps less than 0.1 eV, the layer thicknesses are large and the overlap of electron and hole wavefunctions are small. Thus, the absorption coefficient is too small for useful infrared (IR) detection. Including In in the GaSb introduces strain in he InAs/GaInSb superlattice which shifts the band edges so that small band gaps can be reached in thin layer superlattices. Good absorption at long IR wavelengths is thus achieved.

  8. Magnetic susceptibility, petrofabrics and strain

    NASA Astrophysics Data System (ADS)

    Borradaile, Graham John

    1988-12-01

    Magnetic susceptibility is a non-destructive technique for quantifying the average fabric of a small sample of rock. The interpretation of the magnetic fabric is not always straightforward. However, the principal directions of the magnitude ellipsoid of susceptibility commonly show orientations consistent with the kinematic interpretations of folds, shear zones and other structural features. The directions may correspond with the orientations of strained objects or with the planar-linear mineral orientations. There will usually be multiple mineralogical sources of susceptibility, often involving silicates. If the sources are known, or if the susceptibility can be attributed to a single mineral species, it may be possible to establish a correlation between the strain ellipsoid and the susceptibility ellipsoid. This correlation will be of principal directions in many instances and occasionally there may be a weak correlation of strain magnitudes as well. In other circumstances it may be possible to establish a correlation between changes in susceptibility and the strain. Nevertheless magnetic fabric studies are not routine substitutes for strain analysis. Even where information on strain is not provided, the magnetic fabrics (and subfabrics) yield a measure of the preferred crystallographic orientation or preferred dimensional orientation of the minerals that may be integrated profitably with other petrofabric data. Experimental deformation of certain synthetic aggregates indicates that directions of magnetic susceptibility spin rapidly with advancing strain, especially where the matrix grains undergo crystal-plastic deformation. In certain experiments, simple shear appears to change the intensity of magnetic fabric more effectively than pure shear. Experiments indicate also that the initial anisotropy of a rock-like material is not easily overprinted by deformation whereas field studies are equivocal.

  9. Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway.

    PubMed

    Yang, Wei; Xu, Xiaonan; Li, Yang; Wang, Yingzi; Li, Ming; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.

  10. Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. under organic field conditions.

    PubMed

    Singh, Rakshapal; Soni, Sumit K; Kalra, Alok

    2013-01-01

    Root rot and wilt, caused by a complex involving Fusarium chlamydosporum (Frag. and Cif.) and Ralstonia solanacearum (Smith), are serious diseases affecting the cultivation of Coleus forskohlii, a crop with economic potential as a source of the medicinal compound forskolin. The present 2-year field experiments were conducted with two bioinoculants (a native Pseudomonas monteilii strain and the exotic arbuscular mycorrhizal (AM) fungus Glomus fasciculatum) alone and in combination under organic field conditions in order to evaluate their potential in controlling root rot and wilt. Combined inoculation of P. monteilii with G. fasciculatum significantly increased plant height, plant spread, and number of branches; reduced disease incidence; and increased tuber dry mass of C. forskohlii, compared to vermicompost controls not receiving any bioinoculants. Increase in tuber yields was accompanied by an increase in plant N, P, and K uptake. Co-inoculation of P. monteilii with G. fasciculatum significantly improved the percent AM root colonization and spore numbers retrieved from soil. This suggests P. monteilii to be a mycorrhiza helper bacterium which could be useful in organic agriculture. The forskolin content of tubers was significantly increased by the inoculation treatments of P. monteilii, G. fasciculatum, and P. monteilii + G. fasciculatum.

  11. The expansion of brown rot disease throughout Bolivia: possible role of climate change.

    PubMed

    Castillo, José Antonio; Plata, Giovanna

    2016-05-01

    Bacterial wilt is a devastating plant disease caused by the bacterial pathogen Ralstonia solanacearum species complex and affects different crops. Bacterial wilt infecting potato is also known as brown rot (BR) and is responsible for significant economic losses in potato production, especially in developing countries. In Bolivia, BR affects up to 75% of the potato crop in areas with high incidence and 100% of stored potatoes. The disease has disseminated since its introduction to the country in the mid-1980s mostly through contaminated seed tubers. To avoid this, local farmers multiply seed tubers in highlands because the strain infecting potatoes cannot survive near-freezing temperatures that are typical in the high mountains. Past disease surveys have shown an increase in seed tubers with latent infection in areas at altitudes lower than 3000 m a.s.l. Since global warming is increasing in the Andes Mountains, in this work, we explored the incidence of BR in areas at altitudes above 3000 m a.s.l. Results showed BR presence in the majority of these areas, suggesting a correlation between the increase in disease incidence and the increase in temperature and the number of irregular weather events resulting from climate change. However, it cannot be excluded that the increasing availability of latently infected seed tubers has boosted the spread of BR.

  12. Identification of an antibacterial compound, benzylideneacetone, from Xenorhabdus nematophila against major plant-pathogenic bacteria.

    PubMed

    Ji, Dongjin; Yi, Youngkeun; Kang, Ga-Hwa; Choi, Yong-Hwa; Kim, Pankyung; Baek, Nam-In; Kim, Yonggyun

    2004-10-15

    An entomopathogenic bacterium, Xenorhabdus nematophila, is known to have potent antibiotic activities to maintain monoxenic condition in its insect host for effective pathogenesis and ultimately for optimal development of its nematode symbiont, Steinernema carpocapsae. In this study we assess its antibacterial activity against plant-pathogenic bacteria and identify its unknown antibiotics. The bacterial culture broth had significant antibacterial activity that increased with development of the bacteria and reached its maximum at the stationary growth phase. The antibiotic activities were significant against five plant-pathogenic bacterial strains: Agrobacterium vitis, Pectobacterium carotovorum subsp. atrosepticum, P. carotovorum subsp. carotovorum, Pseudomonas syringae pv. tabaci, and Ralstonia solanacearum. The antibacterial factors were extracted with butanol and fractionated using column chromatography with the eluents of different hydrophobic intensities. Two active antibacterial subfractions were purified, and the higher active fraction was further fractionated and identified as a single compound of benzylideneacetone (trans-4-phenyl-3-buten-2-one). With heat stability, the synthetic compound showed equivalent antibiotic activity and spectrum to the purified compound. This study reports a new antibiotic compound synthesized by X. nematophila, which is a monoterpenoid compound and active against some Gram-negative bacteria.

  13. Optical Strain Measurement System Development

    NASA Technical Reports Server (NTRS)

    Lant, C. T.

    1985-01-01

    Investigations of physical phenomena affecting the durability of SSME components require measurement systems operational in hostile environments. The need for such instrumentation caused the definition and operation of an optical strain measurement system. This optical strain measurement system based on the speckle shift method is being developed. This is a noncontact, automatic method of measuring surface strain in one dimension that corrects for error due to rigid body motion. It provides a gauge length of 1 to 2 mm and allows the region of interest on the test specimen to be mapped point by point. The output is a graphics map of the points inspected on the specimen; data points is stored in quasi-real time. This is the first phase of a multiphase effort in optical strain measurement. The speckle pattern created by the test specimen is interpreted as high order interference fringes resulting from a random diffraction grating, being the natural surface roughness of the specimen. Strain induced on the specimen causes a change in spacing of the surface roughness, which in turn shifts the position of the interference pattern (speckles).

  14. Measuring mine roof bolt strains

    DOEpatents

    Steblay, Bernard J.

    1986-01-01

    A mine roof bolt and a method of measuring the strain in mine roof bolts of this type are disclosed. According to the method, a flat portion on the head of the mine roof bolt is first machined. Next, a hole is drilled radially through the bolt at a predetermined distance from the bolt head. After installation of the mine roof bolt and loading, the strain of the mine roof bolt is measured by generating an ultrasonic pulse at the flat portion. The time of travel of the ultrasonic pulse reflected from the hole is measured. This time of travel is a function of the distance from the flat portion to the hole and increases as the bolt is loaded. Consequently, the time measurement is correlated to the strain in the bolt. Compensation for various factors affecting the travel time are also provided.

  15. Photoacoustic spectroscopy of Entamoeba histolytica strains

    NASA Astrophysics Data System (ADS)

    Acosta-Avalos, D.; Alvarado-Gil, J. J.; Silva, E. F.; Orozco, E.; de Menezes, L. F.; Vargas, H.

    2005-06-01

    Pathogenic and non-pathogenic strains of E. histolytica are studied using photoacoustic spectroscopy. It is shown that the pathogenic strain presents a spectrum similar to that of iron sulfur proteins. The non-pathogenic strain does not show any relevant absorption at the studied wavelength range. The differences observed between the optical absorption spectra of both strains opens the possibility of using photoacoustic spectroscopy as a reliable and simple technique to identify different types of E. histolytica strains.

  16. Characterization of strains of Corynebacterium bovis.

    PubMed Central

    Brooks, B W; Barnum, D A

    1984-01-01

    The biochemical and morphological characteristics of 104 strains of Corynebacterium bovis isolated from bovine milk samples and the C. bovis reference strain were found to be uniform. Valuable criteria for identification were presence of catalase and oxidase, production of acid from glucose and fructose and a requirement for enriched basal media. Six strains of human and three strains of bovine origin were found to be inconsistent with the reference strain. PMID:6722650

  17. Chronic occupational repetitive strain injury.

    PubMed Central

    O'Neil, B. A.; Forsythe, M. E.; Stanish, W. D.

    2001-01-01

    OBJECTIVE: To review common repetitive strain injuries (RSIs) that occur in the workplace, emphasizing diagnosis, treatment, and etiology of these conditions. QUALITY OF EVIDENCE: A MEDLINE search from January 1966 to June 1999 focused on articles published since 1990 because RSIs are relatively new diagnoses. MeSH headings that were explored using the thesaurus included "cumulative trauma disorder," "overuse injury," and "repetitive strain injury." The search was limited to English articles only, and preference was given to randomized controlled trials. MAIN MESSAGE: Repetitive strain injuries result from repeated stress to the body's soft tissue structures including muscles, tendons, and nerves. They often occur in patients who perform repetitive movements either in their jobs or in extracurricular activities. Common RSIs include tendon-related disorders, such as rotator cuff tendonitis, and peripheral nerve entrapment disorders, such as carpal tunnel syndrome. A careful history and physical examination often lead to the diagnosis, but newer imaging techniques, such as magnetic resonance imaging and ultrasound, can help in refractory cases. Conservative management with medication, physiotherapy, or bracing is the mainstay of treatment. Surgery is reserved for cases that do not respond to treatment. CONCLUSION: Repetitive strain injury is common; primary care physicians must establish a diagnosis and, more importantly, its relationship to occupation. Treatment can be offered by family physicians who refer to specialists for cases refractory to conservative management. PMID:11228032

  18. Trials with a Strain Gauge.

    ERIC Educational Resources Information Center

    Auty, Geoff

    1996-01-01

    Describes an attempt to match the goals of the practical demonstration of the use of a strain gauge and the technical applications of science and responding to student questions in early trials, while keeping within the level of electronics in advanced physics. (Author/JRH)

  19. Bacteriocins and novel bacterial strains.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Poultry is thought to be a significant source of Campylobacter in human disease. We evaluated anti-Campylobacter activity among 365 Bacillus and Paenibacillus isolates from poultry. One novel antagonistic Bacillus circulans and three Paenibacillus polymyxa strains were identified and further studi...

  20. Extreme Temperature Strain Measurement System

    DTIC Science & Technology

    1990-08-01

    Electrix Industries, Inc., P.O. Box 306, Lombard, IL 60148, Phone (312) 627-6802 38 Foil Strain gages. ° HiTEC Corp., Nardone Industrial Park...Planer Ltd., CERL-Planer Capacitive Transducer, Windmill Road, Sunbury -on- Thames, Middx., England, Phone: Sunbury 86262-3-4-5 • HiTEC Corp., Nardone

  1. A NEW STRAIN OF TRANSMISSIBLE LEUCEMIA IN FOWLS (STRAIN H).

    PubMed

    Ellermann, V

    1921-03-31

    1. A new strain of fowl leucosis has been transmitted through twelve generations of fowls. 2. An increase in virulence was observed during its passage. This was shown in a shortening of the interval between inoculation and death. The increase in virulence does not affect the number of successful inoculations, which remains approximately constant in from 20 to 40 per cent of the birds employed. 3. As with former strains, the disease manifests itself in various forms; i.e., myeloid and intravascular lymphoid types. A single lymphatic case was observed. 4. In several intravascular cases a diminution in the hemolytic power of the serum was established. This phenomenon was absent in a number of myeloid cases. 5. Active immunization cannot be produced by means of the subcutaneous injection of virulent material. 6. The finding of previous experiments that the virus is filterable has been confirmed. 7. The inoculation of human leucemic material into fowls gave negative results.

  2. Material mechanical characterization method for multiple strains and strain rates

    SciTech Connect

    Erdmand, III, Donald L.; Kunc, Vlastimil; Simunovic, Srdjan; Wang, Yanli

    2016-01-19

    A specimen for measuring a material under multiple strains and strain rates. The specimen including a body having first and second ends and a gage region disposed between the first and second ends, wherein the body has a central, longitudinal axis passing through the first and second ends. The gage region includes a first gage section and a second gage section, wherein the first gage section defines a first cross-sectional area that is defined by a first plane that extends through the first gage section and is perpendicular to the central, longitudinal axis. The second gage section defines a second cross-sectional area that is defined by a second plane that extends through the second gage section and is perpendicular to the central, longitudinal axis and wherein the first cross-sectional area is different in size than the second cross-sectional area.

  3. Strains

    MedlinePlus

    Pulled muscle ... can include: Pain and difficulty moving the injured muscle Discolored and bruised skin Swelling ... if you still have pain. Rest the pulled muscle for at least a day. If possible, keep ...

  4. Modelling to very high strains

    NASA Astrophysics Data System (ADS)

    Bons, P. D.; Jessell, M. W.; Griera, A.; Evans, L. A.; Wilson, C. J. L.

    2009-04-01

    Ductile strains in shear zones often reach extreme values, resulting in typical structures, such as winged porphyroclasts and several types of shear bands. The numerical simulation of the development of such structures has so far been inhibited by the low maximum strains that numerical models can normally achieve. Typical numerical models collapse at shear strains in the order of one to three. We have implemented a number of new functionalities in the numerical platform "Elle" (Jessell et al. 2001), which significantly increases the amount of strain that can be achieved and simultaneously reduces boundary effects that become increasingly disturbing at higher strain. Constant remeshing, while maintaining the polygonal phase regions, is the first step to avoid collapse of the finite-element grid required by finite-element solvers, such as Basil (Houseman et al. 2008). The second step is to apply a grain-growth routine to the boundaries of polygons that represent phase regions. This way, the development of sharp angles is avoided. A second advantage is that phase regions may merge or become separated (boudinage). Such topological changes are normally not possible in finite element deformation codes. The third step is the use of wrapping vertical model boundaries, with which optimal and unchanging model boundaries are maintained for the application of stress or velocity boundary conditions. The fourth step is to shift the model by a random amount in the vertical direction every time step. This way, the fixed horizontal boundary conditions are applied to different material points within the model every time step. Disturbing boundary effects are thus averaged out over the whole model and not localised to e.g. top and bottom of the model. Reduction of boundary effects has the additional advantage that model can be smaller and, therefore, numerically more efficient. Owing to the combination of these existing and new functionalities it is now possible to simulate the

  5. Strain tolerant microfilamentary superconducting wire

    DOEpatents

    Finnemore, Douglas K.; Miller, Theodore A.; Ostenson, Jerome E.; Schwartzkopf, Louis A.; Sanders, Steven C.

    1993-02-23

    A strain tolerant microfilamentary wire capable of carrying superconducting currents is provided comprising a plurality of discontinuous filaments formed from a high temperature superconducting material. The discontinuous filaments have a length at least several orders of magnitude greater than the filament diameter and are sufficiently strong while in an amorphous state to withstand compaction. A normal metal is interposed between and binds the discontinuous filaments to form a normal metal matrix capable of withstanding heat treatment for converting the filaments to a superconducting state. The geometry of the filaments within the normal metal matrix provides substantial filament-to-filament overlap, and the normal metal is sufficiently thin to allow supercurrent transfer between the overlapped discontinuous filaments but is also sufficiently thick to provide strain relief to the filaments.

  6. Virtual strain gage size study

    DOE PAGES

    Reu, Phillip L.

    2015-09-22

    DIC is a non-linear low-pass spatial filtering operation; whether we consider the effect of the subset and shape function, the strain window used in the strain calculation, of other post-processing of the results, each decision will impact the spatial resolution, of the measurement. More fundamentally, the speckle size limits, the spatial resolution by dictating the smallest possible subset. After this decision the processing settings are controlled by the allowable noise level balanced by possible bias errors created by the data filtering. This article describes a process to determine optimum DIC software settings to determine if the peak displacements or strainsmore » are being found.« less

  7. Strain softening in stretched DNA

    PubMed Central

    Luan, Binquan; Aksimentiev, Aleksei

    2010-01-01

    The microscopic mechanics of DNA stretching was characterized using extensive molecular dynamics simulations. By employing an anisotropic pressure control method, realistic force-extension dependences of effectively infinite DNA molecules were obtained. A coexistence of B- and S-DNA domains was observed during the overstretching transition. The simulations revealed that strain softening may occur in the process of stretching torsionally constrained DNA. The latter observation was qualitatively reconciled with available experimental data using a random-field Ising model. PMID:18851334

  8. High Temperature Capacitive Strain Gage

    NASA Technical Reports Server (NTRS)

    Wnuk, Stephen P., Jr.; Wnuk, Stephen P., III; Wnuk, V. P.

    1990-01-01

    Capacitive strain gages designed for measurements in wind tunnels to 2000 F were built and evaluated. Two design approaches were followed. One approach was based on fixed capacitor plates with a movable ground plane inserted between the plates to effect differential capacitive output with strain. The second approach was based on movable capacitor plates suspended between sapphire bearings, housed in a rugged body, and arranged to operate as a differential capacitor. A sapphire bearing gage (1/4 in. diameter x 1 in. in size) was built with a range of 50,000 and a resolution of 200 microstrain. Apparent strain on Rene' 41 was less than + or - 1000 microstrain from room temperature to 2000 F. Three gage models were built from the Ground Plane Differential concept. The first was 1/4 in. square by 1/32 in. high and useable to 700 F. The second was 1/2 in. square by 1/16 in. high and useable to 1440 F. The third, also 1/2 in. square by 1/16 in. high was expected to operate in the 1600 to 2000 F range, but was not tested because time and funding ended.

  9. High temperature capacitive strain gage

    NASA Astrophysics Data System (ADS)

    Wnuk, Stephen P., Jr.; Wnuk, Stephen P., III; Wnuk, V. P.

    1990-01-01

    Capacitive strain gages designed for measurements in wind tunnels to 2000 F were built and evaluated. Two design approaches were followed. One approach was based on fixed capacitor plates with a movable ground plane inserted between the plates to effect differential capacitive output with strain. The second approach was based on movable capacitor plates suspended between sapphire bearings, housed in a rugged body, and arranged to operate as a differential capacitor. A sapphire bearing gage (1/4 in. diameter x 1 in. in size) was built with a range of 50,000 and a resolution of 200 microstrain. Apparent strain on Rene' 41 was less than + or - 1000 microstrain from room temperature to 2000 F. Three gage models were built from the Ground Plane Differential concept. The first was 1/4 in. square by 1/32 in. high and useable to 700 F. The second was 1/2 in. square by 1/16 in. high and useable to 1440 F. The third, also 1/2 in. square by 1/16 in. high was expected to operate in the 1600 to 2000 F range, but was not tested because time and funding ended.

  10. Strain-Detecting Composite Materials

    NASA Technical Reports Server (NTRS)

    Wallace, Terryl A. (Inventor); Smith, Stephen W. (Inventor); Piascik, Robert S. (Inventor); Horne, Michael R. (Inventor); Messick, Peter L. (Inventor); Alexa, Joel A. (Inventor); Glaessgen, Edward H. (Inventor); Hailer, Benjamin T. (Inventor)

    2016-01-01

    A composite material includes a structural material and a shape-memory alloy embedded in the structural material. The shape-memory alloy changes crystallographic phase from austenite to martensite in response to a predefined critical macroscopic average strain of the composite material. In a second embodiment, the composite material includes a plurality of particles of a ferromagnetic shape-memory alloy embedded in the structural material. The ferromagnetic shape-memory alloy changes crystallographic phase from austenite to martensite and changes magnetic phase in response to the predefined critical macroscopic average strain of the composite material. A method of forming a composite material for sensing the predefined critical macroscopic average strain includes providing the shape-memory alloy having an austenite crystallographic phase, changing a size and shape of the shape-memory alloy to thereby form a plurality of particles, and combining the structural material and the particles at a temperature of from about 100-700.degree. C. to form the composite material.

  11. Strains and Sprains Are a Pain

    MedlinePlus

    ... strain at some point. Strains and sprains are common injuries, especially for kids who are very active or ... such as twisting your ankle. This kind of injury is common in sports, but also can happen any time ...

  12. Strain Monitoring of Flexible Structures

    NASA Technical Reports Server (NTRS)

    Litteken, Douglas A.

    2017-01-01

    , such as tensile testing, fatigue testing, and shear testing, but common measurement techniques cannot be used on fabric. Measuring strain in a material and during a test is a critical parameter for an engineer to monitor the structure during the test and correlate to an analytical model. The ability to measure strain in fabric structures is a challenge for NASA. Foil strain gauges, for example, are commonplace on metallic structures testing, but are extremely difficult to interface with a fabric substrate. New strain measuring techniques need to be developed for use with fabric structures. This paper investigates options for measuring strain in fabric structures for both ground testing and in-space structural health monitoring. It evaluates current commercially available options and outlines development work underway to build custom measurement solutions for NASA's fabric structures.

  13. Turbulent Plane Wakes Subjected to Successive Strains

    NASA Technical Reports Server (NTRS)

    Rogers, Michael M.

    2003-01-01

    Six direct numerical simulations of turbulent time-evolving strained plane wakes have been examined to investigate the response of a wake to successive irrotational plane strains of opposite sign. The orientation of the applied strain field has been selected so that the flow is the time-developing analogue of a spatially developing wake evolving in the presence of either a favourable or an adverse streamwise pressure gradient. The magnitude of the applied strain rate a is constant in time t until the total strain e(sup at) reaches about four. At this point, a new simulation is begun with the sign of the applied strain being reversed (the original simulation is continued as well). When the total strain is reduced back to its original value of one, yet another simulation is begun with the sign of the strain being reversed again back to its original sign. This process is done for both initially "favourable" and initially "adverse" strains, providing simulations for each of these strain types from three different initial conditions. The evolution of the wake mean velocity deficit and width is found to be very similar for all the adversely strained cases, with both measures rapidly achieving exponential growth at the rate associated with the cross-stream expansive strain e(sup at). In the "favourably" strained cases, the wake widths approach a constant and the velocity deficits ultimately decay rapidly as e(sup -2at). Although all three of these cases do exhibit the same asymptotic exponential behaviour, the time required to achieve this is longer for the cases that have been previously adversely strained (by at approx. equals 1). These simulations confirm the generality of the conclusions drawn in Rogers (2002) regarding the response of plane wakes to strain. The evolution of strained wakes is not consistent with the predictions of classical self-similar analysis; a more general equilibrium similarity solution is required to describe the results. At least for the cases

  14. Using BEEM To Probe Strains In Semiconductors

    NASA Technical Reports Server (NTRS)

    Bell, L. Douglas; Milliken, Autumn M.; Manion, Stephen J.; Kaiser, William J.

    1996-01-01

    Ballistic-electron-emission microscopy (BEEM) useful in determining strains in semiconductors under some conditions. More specifically, BEEM is variant of scanning tunneling microscopy and sensitive to electronic structure of probed material. In present approach, BEEM used to obtain data on those aspects of variations in electronic structures related to variations in strains. Then by use of mathematical modeling of relationships between electronic structures and strains, variations in strains deduced from BEEM data.

  15. Temperature-Compensating Inactive Strain Gauge

    NASA Technical Reports Server (NTRS)

    Moore, Thomas C., Sr.

    1993-01-01

    Thermal contribution to output of active gauge canceled. High-temperature strain gauges include both active gauge wires sensing strains and inactive gauge wires providing compensation for thermal contributions to gauge readings. Inactive-gauge approach to temperature compensation applicable to commercially available resistance-type strain gauges operating at temperatures up to 700 degrees F and to developmental strain gauges operating at temperatures up to 2,000 degrees F.

  16. Optical Fibers Would Sense Local Strains

    NASA Technical Reports Server (NTRS)

    Egalon, Claudio O.; Rogowski, Robert S.

    1994-01-01

    Proposed fiber-optic transducers measure local strains. Includes lead-in and lead-out lengths producing no changes in phase shifts, plus short sensing length in which phase shift is sensitive to strain. Phase shifts in single-mode fibers vary with strains. In alternative version, multiple portions of optical fiber sensitive to strains characteristic of specific vibrational mode of object. Same principle also used with two-mode fiber.

  17. Strain Rate Effects on Ultimate Strain of Copper

    DTIC Science & Technology

    1979-05-01

    34Dogbone" Specimen Used for Quasi-Static 5 and Intermediate Rate Tests 2 Schematic of Split Hopkinso’v Bar Apparatus 7 3a PETN Filled Tube Specimen...48 Experiment 25 Calculated Stress Components in Copper Cylinder 52 Expanded by PETN 26 Fracture of Explosively Expanded Cylindrical 54 Tube A-la...Record of Shot No. 10 73 A7b Framing Camera Record of Shot No. 10 74 A-8 Strain Versus Time for Copper Tube Expanded 75 by PETN vi

  18. Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains

    PubMed Central

    Gangaiah, Dharanesh; Webb, Kristen M.; Humphreys, Tricia L.; Fortney, Kate R.; Toh, Evelyn; Tai, Albert; Katz, Samantha S.; Pillay, Allan; Chen, Cheng-Yen; Roberts, Sally A.; Munson, Robert S.; Spinola, Stanley M.

    2015-01-01

    Background Although cutaneous ulcers (CU) in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU) and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin? Methodology/Principal Findings To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya) and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin. Conclusions/Significance These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions. PMID:26147869

  19. Antigenic differentiation of classical swine fever vaccinal strain PAV-250 from other strains, including field strains from Mexico.

    PubMed

    Mendoza, Susana; Correa-Giron, Pablo; Aguilera, Edgar; Colmenares, Germán; Torres, Oscar; Cruz, Tonatiuh; Romero, Andres; Hernandez-Baumgarten, Eliseo; Ciprián, Abel

    2007-10-10

    Twenty-nine classical swine fever virus (CSFv) strains were grown in the PK15 or SK6 cell lines. Antigenic differentiation studies were performed using monoclonal antibodies (McAbs), produced at Lelystad (CDI-DLO), The Netherlands. The monoclonals which were classified numerically as monoclonals 2-13. Epitope map patterns that resulted from the reactivity with McAbs were found to be unrelated to the pathogenicity of the viruses studied. Antigenic determinants were recognized by McAbs 5 and 8, were not detected in some Mexican strains; however, sites for McAb 6 were absent in all strains. The PAV-250 vaccine strain was recognized by all MAbs, except by MAb 6. Furthermore, the Chinese C-S vaccine strain was found to be very similar to the GPE(-) vaccine. None of the studied Mexican vaccines or field strains was found to be similar to the PAV-250 vaccine strain.

  20. Effect of strain and strain rate on residual microstructures in copper

    SciTech Connect

    Stevens, M.F.; Follansbee, P.S.

    1986-01-01

    Several specimens of OFE Cu were deformed in compression to study the resulting microstructures at equivalent levels of threshold stress and strain. Equiaxed, diffuse dislocation cells are more persistent in Cu when tested at strain rates exceeding 10/sup 3/ sec/sup -1/. At quasi-static strain rates, dislocation collapse into more distinct, narrow microbands occurs at lower strain levels.

  1. Error quantification in strain mapping methods.

    PubMed

    Guerrero, Elisa; Galindo, Pedro; Yáñez, Andrés; Ben, Teresa; Molina, Sergio I

    2007-10-01

    In this article a method for determining errors of the strain values when applying strain mapping techniques has been devised. This methodology starts with the generation of a thickness/defocus series of simulated high-resolution transmission electron microscopy images of InAsxP1-x/InP heterostructures and the application of geometric phase. To obtain optimal defocusing conditions, a comparison of different defocus values is carried out by the calculation of the strain profile standard deviations among different specimen thicknesses. Finally, based on the analogy of real state strain to a step response, a characterization of strain mapping error near an interface is proposed.

  2. Tests of strain analysis by experimental deformation

    NASA Astrophysics Data System (ADS)

    Borradaile, G. J.; McArthur, J.

    1991-01-01

    The linearisation method and Robin's method of strain analysis of granular materials yield accurate strain estimates for a variety of materials deformed experimentally in pure shear. The breakdown of continuum behaviour at high pore fluid pressures causes the methods to overestimate the strain because they do not take added rigid-body rotation into account. Both methods tolerate some variation in initial shape ratio and some degree of initial preferred orientation at modest strains. Results of tests on polymict sandstone indicate that the lower than average ductility of competent clasts may be balanced against an unfavourable degree of preferred orientation to yield an improved strain estimate.

  3. Radio-Frequency Strain Monitor

    NASA Technical Reports Server (NTRS)

    Heyman, Joseph S.; Rogowski, Robert S.; Holben, Milford S., Jr.

    1988-01-01

    Radio-frequency (RF) strain monitor developed to measure lengths of objects. RF waveguide or cable bonded to structure monitored. Propagation of RF signal along waveguide results in phase shift proportional to length of path traveled. Impedance mismatches placed in RF cable at nodes of structure. Records mismatches and detects overall length of line and lengths of intervals between nodes. Used to detect changes in elements of large structure with single cable. Monitor has potential for many applications, including monitoring stability of such large structures as aircraft, bridges, and buildings in Earthquake zones.

  4. Demonstration test of burner liner strain measurements using resistance strain gages

    NASA Technical Reports Server (NTRS)

    Grant, H. P.; Anderson, W. L.

    1984-01-01

    A demonstration test of burner liner strain measurements using resistance strain gages as well as a feasibility test of an optical speckle technique for strain measurement are presented. The strain gage results are reported. Ten Kanthal A-1 wire strain gages were used for low cycle fatigue strain measurements to 950 K and .002 apparent strain on a JT12D burner can in a high pressure (10 atmospheres) burner test. The procedure for use of the strain gages involved extensive precalibration and postcalibration to correct for cooling rate dependence, drift, and temperature effects. Results were repeatable within + or - .0002 to .0006 strain, with best results during fast decels from 950 K. The results agreed with analytical prediction based on an axisymmetric burner model, and results indicated a non-uniform circumferential distribution of axial strain, suggesting temperature streaking.

  5. Strain engineering of graphene: a review

    NASA Astrophysics Data System (ADS)

    Si, Chen; Sun, Zhimei; Liu, Feng

    2016-02-01

    Graphene has intrigued the science community by many unique properties not found in conventional materials. In particular, it is the strongest two-dimensional material ever measured, being able to sustain reversible tensile elastic strain larger than 20%, which yields an interesting possibility to tune the properties of graphene by strain and thus opens a new field called ``straintronics''. In this article, the current progress in the strain engineering of graphene is reviewed. We first summarize the strain effects on the electronic structure and Raman spectra of graphene. We then highlight the electron-phonon coupling greatly enhanced by the biaxial strain and the strong pseudomagnetic field induced by the non-uniform strain with specific distribution. Finally, the potential application of strain-engineering in the self-assembly of foreign atoms on the graphene surface is also discussed. Given the short history of graphene straintronics research, the current progress has been notable, and many further advances in this field are expected.

  6. Distinguishing bacterial pathogens of potato using a genome-wide microarray approach.

    PubMed

    Aittamaa, M; Somervuo, P; Pirhonen, M; Mattinen, L; Nissinen, R; Auvinen, P; Valkonen, J P T

    2008-09-01

    A set of 9676 probes was designed for the most harmful bacterial pathogens of potato and tested in a microarray format. Gene-specific probes could be designed for all genes of Pectobacterium atrosepticum, c. 50% of the genes of Streptomyces scabies and c. 30% of the genes of Clavibacter michiganensis ssp. sepedonicus utilizing the whole-genome sequence information available. For Streptomyces turgidiscabies, 226 probes were designed according to the sequences of a pathogenicity island containing important virulence genes. In addition, probes were designed for the virulence-associated nip (necrosis-inducing protein) genes of P. atrosepticum, P. carotovorum and Dickeya dadantii and for the intergenic spacer (IGS) sequences of the 16S-23S rRNA gene region. Ralstonia solanacearum was not included in the study, because it is a quarantine organism and is not presently found in Finland, but a few probes were also designed for this species. The probes contained on average 40 target-specific nucleotides and were synthesized on the array in situ, organized as eight sub-arrays with an identical set of probes which could be used for hybridization with different samples. All bacteria were readily distinguished using a single channel system for signal detection. Nearly all of the c. 1000 probes designed for C. michiganensis ssp. sepedonicus, c. 50% and 40% of the c. 4000 probes designed for the genes of S. scabies and P. atrosepticum, respectively, and over 100 probes for S. turgidiscabies showed significant signals only with the respective species. P. atrosepticum, P. carotovorum and Dickeya strains were all detected with 110 common probes. By contrast, the strains of these species were found to differ in their signal profiles. Probes targeting the IGS region and nip genes could be used to place strains of Dickeya to two groups, which correlated with differences in virulence. Taken together, the approach of using a custom-designed, genome-wide microarray provided a robust means

  7. Multiplicative earthquake likelihood models incorporating strain rates

    NASA Astrophysics Data System (ADS)

    Rhoades, D. A.; Christophersen, A.; Gerstenberger, M. C.

    2017-01-01

    SUMMARYWe examine the potential for <span class="hlt">strain</span>-rate variables to improve long-term earthquake likelihood models. We derive a set of multiplicative hybrid earthquake likelihood models in which cell rates in a spatially uniform baseline model are scaled using combinations of covariates derived from earthquake catalogue data, fault data, and <span class="hlt">strain</span>-rates for the New Zealand region. Three components of the <span class="hlt">strain</span> rate estimated from GPS data over the period 1991-2011 are considered: the shear, rotational and dilatational <span class="hlt">strain</span> rates. The hybrid model parameters are optimised for earthquakes of M 5 and greater over the period 1987-2006 and tested on earthquakes from the period 2012-2015, which is independent of the <span class="hlt">strain</span> rate estimates. The shear <span class="hlt">strain</span> rate is overall the most informative individual covariate, as indicated by Molchan error diagrams as well as multiplicative modelling. Most models including <span class="hlt">strain</span> rates are significantly more informative than the best models excluding <span class="hlt">strain</span> rates in both the fitting and testing period. A hybrid that combines the shear and dilatational <span class="hlt">strain</span> rates with a smoothed seismicity covariate is the most informative model in the fitting period, and a simpler model without the dilatational <span class="hlt">strain</span> rate is the most informative in the testing period. These results have implications for probabilistic seismic hazard analysis and can be used to improve the background model component of medium-term and short-term earthquake forecasting models.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.ncbi.nlm.nih.gov/pubmed/25503536','PUBMED'); return false;" href="https://www.ncbi.nlm.nih.gov/pubmed/25503536"><span><span class="hlt">Strain</span> effects on oxygen migration in perovskites.</span></a></p> <p><a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Mayeshiba, Tam; Morgan, Dane</p> <p>2015-01-28</p> <p>Fast oxygen transport materials are necessary for a range of technologies, including efficient and cost-effective solid oxide fuel cells, gas separation membranes, oxygen sensors, chemical looping devices, and memristors. <span class="hlt">Strain</span> is often proposed as a method to enhance the performance of oxygen transport materials, but the magnitude of its effect and its underlying mechanisms are not well-understood, particularly in the widely-used perovskite-structured oxygen conductors. This work reports on an ab initio prediction of <span class="hlt">strain</span> effects on migration energetics for nine perovskite systems of the form LaBO3, where B = [Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Ga]. Biaxial <span class="hlt">strain</span>, as might be easily produced in epitaxial systems, is predicted to lead to approximately linear changes in migration energy. We find that tensile biaxial <span class="hlt">strain</span> reduces the oxygen vacancy migration barrier across the systems studied by an average of 66 meV per percent <span class="hlt">strain</span> for a single selected hop, with a low of 36 and a high of 89 meV decrease in migration barrier per percent <span class="hlt">strain</span> across all systems. The estimated range for the change in migration barrier within each system is ±25 meV per percent <span class="hlt">strain</span> when considering all hops. These results suggest that <span class="hlt">strain</span> can significantly impact transport in these materials, e.g., a 2% tensile <span class="hlt">strain</span> can increase the diffusion coefficient by about three orders of magnitude at 300 K (one order of magnitude at 500 °C or 773 K) for one of the most <span class="hlt">strain</span>-responsive materials calculated here (LaCrO3). We show that a simple elasticity model, which assumes only dilative or compressive <span class="hlt">strain</span> in a cubic environment and a fixed migration volume, can qualitatively but not quantitatively model the <span class="hlt">strain</span> dependence of the migration energy, suggesting that factors not captured by continuum elasticity play a significant role in the <span class="hlt">strain</span> response.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4222745','PMC'); return false;" href="https://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4222745"><span>Distribution of Nontuberculous Mycobacteria <span class="hlt">strains</span></span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p></p> <p>2013-01-01</p> <p>Aim Mycobacteria other than tuberculosis (MOTT) cause increasingly serious infections especially in immunosuppressive patients by direct transmission from the environment or after colonization. However, identification of these species is difficult because of the cost and difficulties in defining to species level. Identification and distribution of these species can help clinician in the choice of treatment. Materials and methods A total of 90 MOTT <span class="hlt">strains</span> obtained from four different centers were included in the study. These <span class="hlt">strains</span> were identified by sequence analysis of 16S rRNA and Hsp65 genetic regions. Results Accordingly, within the 90 MOTT <span class="hlt">strains</span>, 17 different species were identified. In order of frequency, these species were M. gordonea (n = 21), M. abscessus (n = 13), M. lentiflavum (n = 9), M. fortuitum (n = 8), M. intracellulare (n = 6), M. kumamotonense (n = 6), M. neoaurum (n = 5), M. chimaera (n = 5), M. alvei (n = 5), M. peregrinum (n = 3), M. canariasense (n = 3), M. flavescens (n = 1), M. mucogenicum (n = 1), M. chelona (n = 1), M. elephantis (n = 1), M. terrae (n = 1) and M. xenopi (n = 1). Most frequently identified MOTT species according to the geographical origin were as follows: M. abscessus was the most common species either in Istanbul or Malatya regions (n = 6, n = 6, consequently). While M. kumamotonense was the most frequent species isolated from Ankara region (n = 6), M. gordonea was the most common for Samsun region (n = 14). Conclusion Our study revealed that frequency of MOTT varies depending on the number of clinical samples and that frequency of these species were affected by the newly identified species as a result of the use of novel molecular methods. In conclusion, when establishing diagnosis and treatment methods, it is important to know that infections caused by unidentified MOTT species may vary according to the regions in Turkey. The results</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.ncbi.nlm.nih.gov/pubmed/26902321','PUBMED'); return false;" href="https://www.ncbi.nlm.nih.gov/pubmed/26902321"><span>Comparison of <span class="hlt">Strain</span> Rosettes and Digital Image Correlation for Measuring Vertebral Body <span class="hlt">Strain</span>.</span></a></p> <p><a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Gustafson, Hannah; Siegmund, Gunter; Cripton, Peter</p> <p>2016-05-01</p> <p><span class="hlt">Strain</span> gages are commonly used to measure bone <span class="hlt">strain</span>, but only provide <span class="hlt">strain</span> at a single location. Digital image correlation (DIC) is an optical technique that provides the displacement, and therefore <span class="hlt">strain</span>, over an entire region of interest on the bone surface. This study compares vertebral body <span class="hlt">strains</span> measured using <span class="hlt">strain</span> gages and DIC. The anterior surfaces of 15 cadaveric porcine vertebrae were prepared with a <span class="hlt">strain</span> rosette and a speckled paint pattern for DIC. The vertebrae were loaded in compression with a materials testing machine, and two high-resolution cameras were used to image the anterior surface of the bones. The mean noise levels for the <span class="hlt">strain</span> rosette and DIC were 1 με and 24 με, respectively. Bland-Altman analysis was used to compare <span class="hlt">strain</span> from the DIC and rosette (excluding 44% of trials with some evidence of <span class="hlt">strain</span> rosette failure or debonding); the mean difference ± 2 standard deviations (SDs) was -108 με ± 702 με for the minimum (compressive) principal <span class="hlt">strain</span> and -53 με ± 332 με for the maximum (tensile) principal <span class="hlt">strain</span>. Although the DIC has higher noise, it avoids the relatively high risk we observed of <span class="hlt">strain</span> gage debonding. These results can be used to develop guidelines for selecting a method to measure <span class="hlt">strain</span> on bone.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2016AIPC.1723b0001D','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2016AIPC.1723b0001D"><span>Modeling competition between yeast <span class="hlt">strains</span></span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>de Gee, Maarten; van Mourik, Hilda; de Visser, Arjan; Molenaar, Jaap</p> <p>2016-04-01</p> <p>We investigate toxin interference competition between S. cerevisiae colonies grown on a solid medium. In vivo experiments show that the outcome of this competition depends strongly on nutrient availability and cell densities. Here we present a new model for S. cerevisiae colonies, calculating the local height and composition of the colonies. The model simulates yeast colonies that show a good fit to experimental data. Simulations of colonies that start out with a homogeneous mixture of toxin producing and toxin sensitive cells can display remarkable pattern formation, depending on the initial ratio of the <span class="hlt">strains</span>. Simulations in which the toxin producing and toxin sensitive species start at nearby positions clearly show that toxin production is advantageous.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.ncbi.nlm.nih.gov/pubmed/24518385','PUBMED'); return false;" href="https://www.ncbi.nlm.nih.gov/pubmed/24518385"><span>The many shades of prion <span class="hlt">strain</span> adaptation.</span></a></p> <p><a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Baskakov, Ilia V</p> <p>2014-01-01</p> <p>In several recent studies transmissible prion disease was induced in animals by inoculation with recombinant prion protein amyloid fibrils produced in vitro. Serial transmission of amyloid fibrils gave rise to a new class of prion <span class="hlt">strains</span> of synthetic origin. Gradual transformation of disease phenotypes and PrP(Sc) properties was observed during serial transmission of synthetic prions, a process that resembled the phenomenon of prion <span class="hlt">strain</span> adaptation. The current article discusses the remarkable parallels between phenomena of prion <span class="hlt">strain</span> adaptation that accompanies cross-species transmission and the evolution of synthetic prions occurring within the same host. Two alternative mechanisms underlying prion <span class="hlt">strain</span> adaptation and synthetic <span class="hlt">strain</span> evolution are discussed. The current article highlights the complexity of the prion transmission barrier and <span class="hlt">strain</span> adaptation and proposes that the phenomenon of prion adaptation is more common than previously thought.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.dtic.mil/docs/citations/ADA571154','DTIC-ST'); return false;" href="http://www.dtic.mil/docs/citations/ADA571154"><span>SNIT: SNP Identification for <span class="hlt">Strain</span> Typing</span></a></p> <p><a target="_blank" href="https://publicaccess.dtic.mil/psm/api/service/search/search">DTIC Science & Technology</a></p> <p></p> <p>2011-01-01</p> <p>Durkin S, Schneewind O, Nierman WC: Genome sequencing and analysis of Yersina pestis KIM D27, an avirulent <span class="hlt">strain</span> exempt from select agent regulation. PLoS...gener- ated from next-generation sequencing (NGS) data, we selected the recently published Yersinia pestis KIM D27 genome [12]. The Y. pestis D27 <span class="hlt">strain</span>...is a deriva- tive of Y. pestis KIM 10 <span class="hlt">strain</span> (accession no. NC_004088). The Y. pestis KIM D27 draft genome (accession no. ADDC00000000) was generated</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://ntrs.nasa.gov/search.jsp?R=19840000339&hterms=vibrational+modes&qs=N%3D0%26Ntk%3DAll%26Ntx%3Dmode%2Bmatchall%26Ntt%3Dvibrational%2Bmodes','NASA-TRS'); return false;" href="https://ntrs.nasa.gov/search.jsp?R=19840000339&hterms=vibrational+modes&qs=N%3D0%26Ntk%3DAll%26Ntx%3Dmode%2Bmatchall%26Ntt%3Dvibrational%2Bmodes"><span>Deriving <span class="hlt">Strain</span> Modes From Vibrational Tests</span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Young, J. W.; Joanides, J. C.</p> <p>1985-01-01</p> <p>Measurements and theoretical analysis complement each other. Experimental acceleration and <span class="hlt">strain</span> data used to calculate coefficients of low-frequency vibrational modes of object under test. An iterative comparison of experimental and calculated <span class="hlt">strains</span> give modal model of improved accuracy that predicts <span class="hlt">strains</span> under operating conditions. Method useful in fatigue life and reliability analyses of buildings, pumps, engines, vehicles, and other systems subject to vibrations and loud noises during operation.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2016MsT.........54S','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2016MsT.........54S"><span>Construction of an Optical Fiber <span class="hlt">Strain</span> Gauge</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Sulaiman, Najwa</p> <p></p> <p>This project is focused on the construction of an optical fiber <span class="hlt">strain</span> gauge that is based on a <span class="hlt">strain</span> gauge described by Butter and Hocker. Our gauge is designed to generate an interference pattern from the signals carried on two bare single-mode fibers that are fastened to an aluminum cantilever. When the cantilever experiences flexural stress, the interference pattern should change. By observing this change, it is possible to determine the <span class="hlt">strain</span> experienced by the cantilever. I describe the design and construction of our optical fiber <span class="hlt">strain</span> gauge as well as the characterization of different parts of the apparatus.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/912475','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/912475"><span>Measurement of Sorption-Induced <span class="hlt">Strain</span></span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Eric P. Robertson; Richard L. Christiansen</p> <p>2005-05-01</p> <p><span class="hlt">Strain</span> caused by the adsorption of gases was measured in samples of subbituminous coal from the Powder River basin of Wyoming, U.S.A. and high-volatile bituminous coal from east-central Utah, U.S.A. using an apparatus developed jointly at the Idaho National Laboratory (Idaho Falls, Idaho, U.S.A.) and Colorado School of Mines (Golden, Colorado, U.S.A.). The apparatus can be used to measure <span class="hlt">strain</span> on multiple small coal samples based on the optical detection of the longitudinal <span class="hlt">strain</span> instead of the more common usage of <span class="hlt">strain</span> gauges, which require larger samples and longer equilibration times. With this apparatus, we showed that the swelling and shrinkage processes were reversible and that accurate <span class="hlt">strain</span> data could be obtained in a shortened amount of time. A suite of <span class="hlt">strain</span> curves was generated for these coals using gases that included carbon dioxide, nitrogen, methane, helium, and various mixtures of these gases. A Langmuir-type equation was applied to satisfactorily model the <span class="hlt">strain</span> data obtained for pure gases. The sorption-induced <span class="hlt">strain</span> measured in the subbituminous coal was larger than the high-volatile bituminous coal for all gases tested over the range of pressures used in the experimentation, with the CO2-induced <span class="hlt">strain</span> for the subbituminous coal over twice as great at the bituminous coal.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://hdl.handle.net/2060/20160000697','NASA-TRS'); return false;" href="http://hdl.handle.net/2060/20160000697"><span>Acceleration and Velocity Sensing from Measured <span class="hlt">Strain</span></span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Pak, Chan-Gi; Truax, Roger</p> <p>2016-01-01</p> <p>A simple approach for computing acceleration and velocity of a structure from the <span class="hlt">strain</span> is proposed in this study. First, deflection and slope of the structure are computed from the <span class="hlt">strain</span> using a two-step theory. Frequencies of the structure are computed from the time histories of <span class="hlt">strain</span> using a parameter estimation technique together with an Autoregressive Moving Average model. From deflection, slope, and frequencies of the structure, acceleration and velocity of the structure can be obtained using the proposed approach. shape sensing, fiber optic <span class="hlt">strain</span> sensor, system equivalent reduction and expansion process.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.osti.gov/scitech/biblio/20976649','SCIGOV-STC'); return false;" href="https://www.osti.gov/scitech/biblio/20976649"><span><span class="hlt">Strain</span> accommodation in inelastic deformation of glasses</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Murali, P.; Ramamurty, U.; Shenoy, Vijay B.</p> <p>2007-01-01</p> <p>Motivated by recent experiments on metallic glasses, we examine the micromechanisms of <span class="hlt">strain</span> accommodation including crystallization and void formation during inelastic deformation of glasses by employing molecular statics simulations. Our atomistic simulations with Lennard-Jones-like potentials suggests that a softer short range interaction between atoms favors crystallization. Compressive hydrostatic <span class="hlt">strain</span> in the presence of a shear <span class="hlt">strain</span> promotes crystallization whereas a tensile hydrostatic <span class="hlt">strain</span> is found to induce voids. The deformation subsequent to the onset of crystallization includes partial reamorphization and recrystallization, suggesting important atomistic mechanisms of plastic dissipation in glasses.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/366503','SCIGOV-STC'); return false;" href="http://www.osti.gov/scitech/servlets/purl/366503"><span>Investigation of a noncontact <span class="hlt">strain</span> measurement technique</span></a></p> <p><a target="_blank" href="http://www.osti.gov/scitech">SciTech Connect</a></p> <p>Damiano, B.; Talarico, L.J.</p> <p>1996-05-01</p> <p>The goal of this project was to investigate the feasibility of a new noncontact technique for directly and continuously monitoring peak <span class="hlt">strain</span> in rotating components. The technique utilizes the unique <span class="hlt">strain</span>-sensitive magnetic material properties of transformation Induced Plasticity (TRIP) steel alloys to measure <span class="hlt">strain</span>. These alloys are weakly magnetic when unstrained but become strongly ferromagnetic after mechanical deformation. A computer study was performed to determine whether the <span class="hlt">strain</span>-induced change in the magnetic material properties of a TRIP steel gage bonded to a rotating component would cause significant perturbations in the magnetic flux of a stationary electromagnet. The effects of <span class="hlt">strain</span> level, distance between the rotating component and the stationary electromagnet, and motion-induced eddy currents on flux perturbation magnitude were investigated. The calculated results indicate that a TRIP steel <span class="hlt">strain</span> sensing element can cause a significant perturbation in the magnetic flux of a stationary electromagnet. The magnetic flux perturbation magnitude was found to be inversely proportional to the distance between the magnet face and the TRIP steel element and directly proportional to the TRIP steel <span class="hlt">strain</span> level. The effect of motion-induced eddy currents on the magnetic flux was found to be negligible. It appears that the technique can be successfully applied to measure peak <span class="hlt">strain</span> in rotating components; however, the sensitivity of the magnetic flux perturbation magnitude to the distance between the <span class="hlt">strain</span> sensing element and the electromagnet may require making an independent proximity measurement.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.ncbi.nlm.nih.gov/pubmed/7453585','PUBMED'); return false;" href="https://www.ncbi.nlm.nih.gov/pubmed/7453585"><span>[Antigenic relationships between Debaryomyces <span class="hlt">strains</span> (author's transl)].</span></a></p> <p><a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Aksoycan, N</p> <p>1980-01-01</p> <p>The results of the agglutinations between homologous and heterologous Debaryomyces <span class="hlt">strains</span> and their agglutinating sera are shown in table I. According to these findings, D. hansenii and D. marama are antigenically different from other Debaryomyces <span class="hlt">strains</span> in this genus. In a previous study Aksoycan et al. have shown a common antigenic factor between D. hansenii, D. marama <span class="hlt">strains</span> and Salmonella 0:7 antigen. This factor was not present in other six <span class="hlt">strains</span> of Debaryomyces. These results also show that D. tamarii does not have any antigenic relationship with the other seven species of Debaryomyces in this genus.</p> </li> </ol> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li class="active"><span>24</span></li> <li><a href="#" onclick='return showDiv("page_25");'>25</a></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div><!-- col-sm-12 --> </div><!-- row --> </div><!-- page_24 --> <div id="page_25" class="hiddenDiv"> <div class="row"> <div class="col-sm-12"> <div class="pull-right"> <ul class="pagination"> <li><a href="#" onclick='return showDiv("page_1");'>«</a></li> <li><a href="#" onclick='return showDiv("page_21");'>21</a></li> <li><a href="#" onclick='return showDiv("page_22");'>22</a></li> <li><a href="#" onclick='return showDiv("page_23");'>23</a></li> <li><a href="#" onclick='return showDiv("page_24");'>24</a></li> <li class="active"><span>25</span></li> <li><a href="#" onclick='return showDiv("page_25");'>»</a></li> </ul> </div> </div> </div> <div class="row"> <div class="col-sm-12"> <ol class="result-class" start="481"> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2012APS..MARV27001L','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2012APS..MARV27001L"><span>Making Novel Materials Using <span class="hlt">Strain</span> in Nanomembranes</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Lagally, Max G.</p> <p>2012-02-01</p> <p>The controlled introduction of <span class="hlt">strain</span> in materials offers an important degree of freedom for fundamental studies of materials as well as advanced device engineering. <span class="hlt">Strain</span> in a crystalline solid modifies the lattice constants and reduces the crystal symmetry. Because <span class="hlt">strain</span> energy is proportional to thickness, a free-standing crystalline thin sheet, which we call a nanomembrane (NM), can be <span class="hlt">strained</span> to a greater degree that a bulk material with the same surface area. I show the use of nanomembrane <span class="hlt">strain</span> engineering to make defect-free single crystals that cannot be grown any other way, and materials with <span class="hlt">strain</span> symmetries that they do not have naturally, in both cases alloys of Si and Ge. <span class="hlt">Strain</span> in NMs causes significant shifts in energy band edges, splitting of degenerate states, and changes in effective masses. These effects can be used to produce a desired band offset between different materials, to increase carrier mobility, and to change relative energy positions of valleys. In the latter respect, through the use of NMs it has recently become possible, using tensile <span class="hlt">strain</span>, to make Ge direct-bandgap and light emitting at room temperature. Periodic local stress can produce <span class="hlt">strain</span> superlattices and thus single-element heterojunctions. Work performed with the Roberto Paiella, Mark Eriksson, Feng Liu, and Irena Knezevic research groups.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://www.osti.gov/scitech/servlets/purl/867015','DOE-PATENT-XML'); return false;" href="http://www.osti.gov/scitech/servlets/purl/867015"><span>Optical fiber sensor technique for <span class="hlt">strain</span> measurement</span></a></p> <p><a target="_blank" href="http://www.osti.gov/doepatents">DOEpatents</a></p> <p>Butler, Michael A.; Ginley, David S.</p> <p>1989-01-01</p> <p>Laser light from a common source is split and conveyed through two similar optical fibers and emitted at their respective ends to form an interference pattern, one of the optical fibers having a portion thereof subjected to a <span class="hlt">strain</span>. Changes in the <span class="hlt">strain</span> cause changes in the optical path length of the <span class="hlt">strain</span> fiber, and generate corresponding changes in the interference pattern. The interference pattern is received and transduced into signals representative of fringe shifts corresponding to changes in the <span class="hlt">strain</span> experienced by the <span class="hlt">strained</span> one of the optical fibers. These signals are then processed to evaluate <span class="hlt">strain</span> as a function of time, typical examples of the application of the apparatus including electrodeposition of a metallic film on a conductive surface provided on the outside of the optical fiber being <span class="hlt">strained</span>, so that <span class="hlt">strains</span> generated in the optical fiber during the course of the electrodeposition are measurable as a function of time. In one aspect of the invention, signals relating to the fringe shift are stored for subsequent processing and analysis, whereas in another aspect of the invention the signals are processed for real-time display of the <span class="hlt">strain</span> changes under study.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2013AcMSn..29..543X','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2013AcMSn..29..543X"><span>Control of surface wettability via <span class="hlt">strain</span> engineering</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Xiong, Wei; Liu, Jefferson Zhe; Zhang, Zhi-Liang; Zhen, Quan-Shui</p> <p>2013-08-01</p> <p>Reversible control of surface wettability has wide applications in lab-on-chip systems, tunable optical lenses, and microfluidic tools. Using a graphene sheet as a sample material and molecular dynamic simulations, we demonstrate that <span class="hlt">strain</span> engineering can serve as an effective way to control the surface wettability. The contact angles θ of water droplets on a graphene vary from 72.5° to 106° under biaxial <span class="hlt">strains</span> ranging from -10% to 10% that are applied on the graphene layer. For an intrinsic hydrophilic surface (at zero <span class="hlt">strain</span>), the variation of θ upon the applied <span class="hlt">strains</span> is more sensitive, i.e., from 0° to 74.8°. Overall the cosines of the contact angles exhibit a linear relation with respect to the <span class="hlt">strains</span>. In light of the inherent dependence of the contact angle on liquid-solid interfacial energy, we develop an analytic model to show the cos θ as a linear function of the adsorption energy E ads of a single water molecule over the substrate surface. This model agrees with our molecular dynamic results very well. Together with the linear dependence of E ads on biaxial <span class="hlt">strains</span>, we can thus understand the effect of <span class="hlt">strains</span> on the surface wettability. Thanks to the ease of reversibly applying mechanical <span class="hlt">strains</span> in micro/nano-electromechanical systems, we believe that <span class="hlt">strain</span> engineering can be a promising means to achieve the reversibly control of surface wettability.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://hdl.handle.net/2060/19840004375','NASA-TRS'); return false;" href="http://hdl.handle.net/2060/19840004375"><span>Inflatable device for installing <span class="hlt">strain</span> gage bridges</span></a></p> <p><a target="_blank" href="http://ntrs.nasa.gov/search.jsp">NASA Technical Reports Server (NTRS)</a></p> <p>Cook, C. E.; Smith, G. E.; Monaghan, R. C. (Inventor)</p> <p>1983-01-01</p> <p>Methods and devices for installing in a tubular shaft multiple <span class="hlt">strain</span> gages are disclosed with focus on a method and a device for pneumatically forcing <span class="hlt">strain</span> gages into seated engagement with the internal surfaces of a tubular shaft in an installation of multiple <span class="hlt">strain</span> gages in a tubular shaft. The <span class="hlt">strain</span> gages or other electron devices are seated in a template-like component which is wrapped about a pneumatically expansible body. The component is inserted into a shaft and the body is pneumatically expanded after a suitable adhesive was applied to the surfaces.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2002SPIE.4700..304A','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2002SPIE.4700..304A"><span>Microminiature temperature-compensated magnetoelastic <span class="hlt">strain</span> gauge</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Arms, Steven W.; Townsend, Christopher P.</p> <p>2002-07-01</p> <p>Our objective was to demonstrate a microminiature magnetoelastic <span class="hlt">strain</span> gauge that provides both <span class="hlt">strain</span> and temperature signals without additional sensors. Iron based magnetoelastic materials were embedded within superelastic nickel/titanium (NiTi) tubing. NiTi stress was transferred to the ferrite, causing a permeability change sensed by a tiny coil. The coil/bridge was excited (70 KHz AC), synchronously demodulated, and amplified to produce a voltage output proportional to coil/ferrite impedance. A DC voltage was also applied and separately conditioned to provide an output proportional to coil resistance; this signal was used to provide thermal compensation. Controlled <span class="hlt">strains</span> were applied and 6 Hz cyclic outputs recorded simultaneously from the magnetoelastic <span class="hlt">strain</span> gauge and conventional foil <span class="hlt">strain</span> gauges. The magnetoelastic <span class="hlt">strain</span> gauge tracked the foil gauge with minimal hysteresis and good linearity over 600 microstrain; repeatability was approximately 1.5 microstrain. The magnetoelastic <span class="hlt">strain</span> gauge's gauge factor was computed from delta inductance/original inductance under static <span class="hlt">strain</span> conditions. Temperatures of 25-140 deg C resulted in an uncompensated shift of 15 microstrain/deg C, and compensated shift of 1.0 microstrain/deg C. A sensitive micro-magnetoelastic <span class="hlt">strain</span> gauge was demonstrated using the same sensor to detect stress and temperature with no moving parts, high gauge factor, and good thermal stability.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('http://adsabs.harvard.edu/abs/2014SPIE.9061E..31R','NASAADS'); return false;" href="http://adsabs.harvard.edu/abs/2014SPIE.9061E..31R"><span>Distributed <span class="hlt">strain</span> monitoring for bridges: temperature effects</span></a></p> <p><a target="_blank" href="http://adsabs.harvard.edu/abstract_service.html">NASA Astrophysics Data System (ADS)</a></p> <p>Regier, Ryan; Hoult, Neil A.</p> <p>2014-03-01</p> <p>To better manage infrastructure assets as they reach the end of their service lives, quantitative data is required to better assess structural behavior and allow for more informed decision making. Distributed fiber optic <span class="hlt">strain</span> sensors are one sensing technology that could provide comprehensive data for use in structural assessments as these systems potentially allow for <span class="hlt">strain</span> to be measured with the same accuracy and gage lengths as conventional <span class="hlt">strain</span> sensors. However, as with many sensor technologies, temperature can play an important role in terms of both the structure's and sensor's performance. To investigate this issue a fiber optic distributed <span class="hlt">strain</span> sensor system was installed on a section of a two span reinforced concrete bridge on the TransCanada Highway. <span class="hlt">Strain</span> data was acquired several times a day as well as over the course of several months to explore the effects of changing temperature on the data. The results show that the <span class="hlt">strain</span> measurements are affected by the bridge behavior as a whole. The <span class="hlt">strain</span> measurements due to temperature are compared to <span class="hlt">strain</span> measurements that were taken during a load test on the bridge. The results show that even a small change in temperature can produce crack width and <span class="hlt">strain</span> changes similar to those due to a fully loaded transport truck. Future directions for research in this area are outlined.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4644690','PMC'); return false;" href="https://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=4644690"><span>Skeletal muscle tensile <span class="hlt">strain</span> dependence: hyperviscoelastic nonlinearity</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Wheatley, Benjamin B; Morrow, Duane A; Odegard, Gregory M; Kaufman, Kenton R; Donahue, Tammy L Haut</p> <p>2015-01-01</p> <p>Introduction Computational modeling of skeletal muscle requires characterization at the tissue level. While most skeletal muscle studies focus on hyperelasticity, the goal of this study was to examine and model the nonlinear behavior of both time-independent and time-dependent properties of skeletal muscle as a function of <span class="hlt">strain</span>. Materials and Methods Nine tibialis anterior muscles from New Zealand White rabbits were subject to five consecutive stress relaxation cycles of roughly 3% <span class="hlt">strain</span>. Individual relaxation steps were fit with a three-term linear Prony series. Prony series coefficients and relaxation ratio were assessed for <span class="hlt">strain</span> dependence using a general linear statistical model. A fully nonlinear constitutive model was employed to capture the <span class="hlt">strain</span> dependence of both the viscoelastic and instantaneous components. Results Instantaneous modulus (p<0.0005) and mid-range relaxation (p<0.0005) increased significantly with <span class="hlt">strain</span> level, while relaxation at longer time periods decreased with <span class="hlt">strain</span> (p<0.0005). Time constants and overall relaxation ratio did not change with <span class="hlt">strain</span> level (p>0.1). Additionally, the fully nonlinear hyperviscoelastic constitutive model provided an excellent fit to experimental data, while other models which included linear components failed to capture muscle function as accurately. Conclusions Material properties of skeletal muscle are <span class="hlt">strain</span>-dependent at the tissue level. This <span class="hlt">strain</span> dependence can be included in computational models of skeletal muscle performance with a fully nonlinear hyperviscoelastic model. PMID:26409235</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2951977','PMC'); return false;" href="https://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2951977"><span>Prion <span class="hlt">strain</span> interactions are highly selective</span></a></p> <p><a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pmc">PubMed Central</a></p> <p>Nilsson, K. Peter R.; Joshi-Barr, Shivanjali; Winson, Olivia; Sigurdson, Christina J.</p> <p>2010-01-01</p> <p>Various misfolded and aggregated neuronal proteins commonly co-exist in neurodegenerative disease, but whether the proteins co-aggregate and alter the disease pathogenesis is unclear. Here we used mixtures of distinct prion <span class="hlt">strains</span>, which are believed to differ in conformation, to test the hypothesis that two different aggregates interact and change the disease in vivo. We tracked two prion <span class="hlt">strains</span> in mice histopathologically and biochemically, as well as by spectral analysis of plaque-bound polythiophene acetic acid (PTAA), a conformation-sensitive fluorescent amyloid ligand. We found that prion <span class="hlt">strains</span> interacted in a highly selective and <span class="hlt">strain</span>-specific manner, with either (i) no interaction, (ii) hybrid plaque formation, or (iii) blockage of one <span class="hlt">strain</span> by a second (interference). The hybrid plaques were maintained upon further passage in vivo and each <span class="hlt">strain</span> seemed to maintain its original conformational properties, suggesting that one <span class="hlt">strain</span> served only as a scaffold for aggregation of the second <span class="hlt">strain</span>. These findings not only further our understanding of prion <span class="hlt">strain</span> interactions, but also directly demonstrate interactions that may occur in other protein aggregate mixtures. PMID:20826672</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.ncbi.nlm.nih.gov/pubmed/9749560','PUBMED'); return false;" href="https://www.ncbi.nlm.nih.gov/pubmed/9749560"><span><span class="hlt">Strain</span> amplification in the bone mechanosensory system.</span></a></p> <p><a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Cowin, S C; Weinbaum, S</p> <p>1998-09-01</p> <p>This article discusses the potential mechanisms by which the <span class="hlt">strain</span> induced at the membrane of an osteocyte may be amplified from the <span class="hlt">strain</span> experienced by the whole bone due to mechanical loading. These mechanisms address the question of how these mechanical load-induced small <span class="hlt">strains</span> of (typically) about 0.1% (but up to 0.5%) applied to a whole bone are amplified to <span class="hlt">strains</span> of 1% or larger at the membrane of the osteocyte buried in its lacuna in the bone matrix. The answer to this question is an important link in the mechanosensory system in bone and in relating in vitro cell studies to in vivo cellular response.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.ncbi.nlm.nih.gov/pubmed/10878089','PUBMED'); return false;" href="https://www.ncbi.nlm.nih.gov/pubmed/10878089"><span>Surveillance of rotavirus <span class="hlt">strains</span> in the United States: identification of unusual <span class="hlt">strains</span>. The National Rotavirus <span class="hlt">Strain</span> Surveillance System collaborating laboratories.</span></a></p> <p><a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Griffin, D D; Kirkwood, C D; Parashar, U D; Woods, P A; Bresee, J S; Glass, R I; Gentsch, J R</p> <p>2000-07-01</p> <p>Rotavirus <span class="hlt">strains</span> from 964 fecal specimens collected from children at 11 U.S. hospital laboratories from November 1997 to March 1998 and from samples collected at 12 laboratories from November 1998 to March 1999 were typed for G and P proteins. Serotype G1 was the predominant serotype in 1997-1998 (88%), followed by G2 (6.2%), G9 (3.3%), and G3 (1.5%). This pattern was similar to that seen in 1998-1999: G1 (79%), G2 (15%), G9 (3.0%), G4 (1.6%), and G3 (0.3%). Novel P[9] <span class="hlt">strains</span> were identified in both seasons, and analysis of a 364-nucleotide fragment from gene segment 4 of one of the <span class="hlt">strains</span> demonstrated 97.3% nucleotide identity with the prototype P3[9],G3 <span class="hlt">strain</span>, AU1, isolated in Japan. This is the first report of a human AU1-like <span class="hlt">strain</span> in the United States. These results reinforce our initial findings that serotype G9 persists in the United States but has not become a predominant <span class="hlt">strain</span> and that the common serotypes G1 to G4 account for almost 90% of <span class="hlt">strains</span> in circulation. Other uncommon <span class="hlt">strains</span> exist in the United States but may have been overlooked before because of their low prevalence and the use of inadequate diagnostic tools.</p> </li> <li> <p><a target="_blank" onclick="trackOutboundLink('https://www.ncbi.nlm.nih.gov/pubmed/26080741','PUBMED'); return false;" href="https://www.ncbi.nlm.nih.gov/pubmed/26080741"><span>Structure-Activity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen.</span></a></p> <p><a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed">PubMed</a></p> <p>Farag, Mohamed A; Al-Mahdy, Dalia A; Salah El Dine, Riham; Fahmy, Sherifa; Yassin, Aymen; Porzel, Andrea; Brandt, Wolfgang</p> <p>2015-06-01</p> <p>Bacterial wilts of potato, tomato, pepper, and or eggplant caused by <span class="hlt">Ralstonia</span> <span class="hlt">solanacearum</span> are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. <span class="hlt">solanacearum</span> derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc-zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity-guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5-dihydroxy-4-methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed signif