Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Xylella fastidiosa subsp pauca causes citrus variegat...

Evaluation of assembling methods on determination of whole genome sequence of Xylella fastidiosa blueberry bacterial leaf scorch strain

USDA-ARS?s Scientific Manuscript database

Blueberry bacterial leaf scorch (BBLS) disease, a threat to blueberry production in the Southern USA and potentially elsewhere, is caused by Xylella fastidiosa. Efficient control of BBLS requires knowledge of the pathogen. However, this is challenging because Xylella fastidiosa is difficult to cultu...

Characterization of a diffusible signaling factor from Xylella fastidiosa.

PubMed

Beaulieu, Ellen D; Ionescu, Michael; Chatterjee, Subhadeep; Yokota, Kenji; Trauner, Dirk; Lindow, Steven

2013-01-08

Cell-cell signaling in Xylella fastidiosa has been implicated in the coordination of traits enabling colonization in plant hosts as well as insect vectors. This cell density-dependent signaling has been attributed to a diffusible signaling factor (DSF) produced by the DSF synthase RpfF. DSF produced by related bacterial species are unsaturated fatty acids, but that of X. fastidiosa was thought to be different from those of other taxa. We describe here the isolation and characterization of an X. fastidiosa DSF (XfDSF) as 2(Z)-tetradecenoic acid. This compound was isolated both from recombinant Erwinia herbicola expressing X. fastidiosa rpfF and from an X. fastidiosa rpfC deletion mutant that overproduces DSF. Since an rpfF mutant is impaired in biofilm formation and underexpresses the hemagglutinin-like protein-encoding genes hxfA and hxfB, we demonstrate that these traits can be restored by ca. 0.5 µM XfDSF but not by myristic acid, the fully saturated tetradecenoic acid. A phoA-based X. fastidiosa biosensor that assesses DSF-dependent expression of hxfA or hxfB revealed a high level of molecular specificity of DSF signaling. X. fastidiosa causes diseases in many important plants, including grape, where it incites Pierce's disease. Virulence of X. fastidiosa for grape is coordinated by cell-cell signaling molecules, designated DSF (Diffusible Signaling Factor). Mutants blocked in DSF production are hypervirulent for grape, suggesting that virulence is suppressed upon DSF accumulation and that disease could be controlled by artificial elevation of the DSF level in plants. In this work, we describe the isolation of the DSF produced by X. fastidiosa and the verification of its biological activity as an antivirulence factor. We also have developed X. fastidiosa DSF biosensors to evaluate the specificity of cell-cell signaling to be investigated.

Xylella fastidiosa Afimbrial Adhesins Mediate Cell Transmission to Plants by Leafhopper Vectors▿

PubMed Central

Killiny, Nabil; Almeida, Rodrigo P. P.

2009-01-01

The interactions between the economically important plant-pathogenic bacterium Xylella fastidiosa and its leafhopper vectors are poorly characterized. We used different approaches to determine how X. fastidiosa cells interact with the cuticular surface of the foreguts of vectors. We demonstrate that X. fastidiosa binds to different polysaccharides with various affinities and that these interactions are mediated by cell surface carbohydrate-binding proteins. In addition, competition assays showed that N-acetylglucosamine inhibits bacterial adhesion to vector foregut extracts and intact wings, demonstrating that attachment to leafhopper surfaces is affected in the presence of specific polysaccharides. In vitro experiments with several X. fastidiosa knockout mutants indicated that hemagglutinin-like proteins are associated with cell adhesion to polysaccharides. These results were confirmed with biological experiments in which hemagglutinin-like protein mutants were transmitted to plants by vectors at lower rates than that of the wild type. Furthermore, although these mutants were defective in adhesion to the cuticle of vectors, their growth rate once attached to leafhoppers was similar to that of the wild type, suggesting that these proteins are important for initial adhesion of X. fastidiosa to leafhoppers. We propose that X. fastidiosa colonization of leafhopper vectors is a complex, stepwise process similar to the formation of biofilms on surfaces. PMID:19011051

Identification and characterization of plasmids from Xylella fastidiosa using next generation sequencing analyses

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa causes diseases in many economically important crops, e.g. Pierce’s disease (PD) of grapevines and blueberry bacterial leaf scorch (BBLS) disease in the U.S. Biological research on X. fastidiosa has been difficult due to its nutritional fastidiousness. Genomic research provides a ...

Temperature dependent RNA metabolism in Xylella fastidiosa during cold stress and grapevine infection

USDA-ARS?s Scientific Manuscript database

Re-occurrence of Pierce’s disease of grapes, caused by Xylella fastidiosa, is known to be influenced by environmental factors, particularly cold temperatures during overwintering. Grapevines in colder regions are often cured of X. fastidiosa infection over the winter season, depending on cultivar, t...

Xylella fastidiosa in rabbiteye blueberry: A newly studied host of an old foe

USDA-ARS?s Scientific Manuscript database

The bacterium Xylella fastidiosa causes a number of plant diseases, including bacterial leaf scorch of southern highbush blueberry. In Louisiana, X. fastidiosa has been detected in rabbiteye blueberry orchards, and we wanted to know if it affected yield in rabbiteye blueberry plants. We detected X...

Evaluation of olive as a host of Xylella fastidiosa and associated sharpshooter vectors

USDA-ARS?s Scientific Manuscript database

Olive (Olea europaea L.) trees exhibiting leaf scorch and/or branch dieback symptoms in California were surveyed for the xylem-limited, fastidious bacterium Xylella fastidiosa. Only ~17% of diseased trees tested positive for X. fastidiosa by PCR, and disease symptoms could not be attributed to X. fa...

Network analysis reveals why Xylella fastidiosa will persist in Europe.

PubMed

Strona, Giovanni; Carstens, Corrie Jacobien; Beck, Pieter S A

2017-03-06

The insect vector borne bacterium Xylella fastidiosa was first detected in olive trees in Southern Italy in 2013, and identified as the main culprit behind the 'olive quick decline syndrome'. Since then, the disease has spread rapidly through Italy's main olive oil producing region. The epidemiology of the outbreak is largely unstudied, with the list of X. fastidiosa hosts and vectors in Europe likely incomplete, and the role humans play in dispersal unknown. These knowledge gaps have led to management strategies based on general assumptions that require, among others, local vector control and, in certain areas, the destruction of infected plants and healthy ones around them in an attempt to eradicate or halt the spreading pest. Here we show that, regardless of epidemiological uncertainties, the mere distribution of olive orchards in Southern Italy makes the chances of eradicating X. fastidiosa from the region extremely slim. Our results imply that Southern Italy is becoming a reservoir for X. fastidiosa. As a consequence, management strategies should keep the prevalence of X. fastidiosa in the region as low as possible, primarily through vector control, lest the pathogen, that has also been detected in southern France and the island of Mallorca (Spain), continues spreading through Italy and Europe.

Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Prophage in B. pseudomallei 1026b

DTIC Science & Technology

2004-06-01

identification of several new virulence gene candidates. In particular, K96243 harbors multiple genomic islands with relatively low GC contents...differences were observed. Prophage-encoded virulence factors in other bacterial species have been described (5), and it was of interest to see if gene ... Xylella fastidiosa (11, 16, 17). The genomic sequencing results for multiple strains of Streptococcus and Xylella suggest that different disease

Conjugative plasmid transfer in Xylella fastidiosa is dependent on tra and trb operon functions

USDA-ARS?s Scientific Manuscript database

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer and recombination, leading to diversity between strains and the categorization of X. fastidiosa into multiple subspecies. Although natural transformation is shown to occur at high rates in X. fa...

Identification of novel secreted virulence factors from Xylella fastidiosa using a TRV expression system

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a bacterium that causes leaf scorch diseases of agriculturally important crops including grapevines and almonds. Little is known about virulence factors that are necessary for X. fastidiosa to grow and cause disease in the xylem vessels of a plant host. Any protein secreted by ...

The endophyte Curtobacterium flaccumfaciens reduces symptoms caused by Xylella fastidiosa in Catharanthus roseus.

PubMed

Lacava, Paulo Teixeira; Li, Wenbin; Araújo, Welington Luiz; Azevedo, João Lúcio; Hartung, John Stephen

2007-10-01

Citrus variegated chlorosis (CVC) is a disease of the sweet orange [Citrus sinensis (L.)], which is caused by Xylella fastidiosa subsp. pauca, a phytopathogenic bacterium that has been shown to infect all sweet orange cultivars. Sweet orange trees have been occasionally observed to be infected by Xylella fastidiosa without evidencing severe disease symptoms, whereas other trees in the same grove may exhibit severe disease symptoms. The principal endophytic bacterial species isolated from such CVC-asymptomatic citrus plants is Curtobacterium flaccumfaciens. The Madagascar periwinkle [Citrus sinensis (L.)] is a model plant which has been used to study X. fastidiosa in greenhouse environments. In order to characterize the interactions of X. fastidiosa and C. flaccumfaciens, periwinkle plants were inoculated separately with C. flaccumfaciens, X. fastidiosa, and both bacteria together. The number of flowers produced by the plants, the heights of the plants, and the exhibited disease symptoms were evaluated. PCR-primers for C. flaccumfaciens were designed in order to verify the presence of this endophytic bacterium in plant tissue, and to complement an existing assay for X. fastidiosa. These primers were capable of detecting C. flaccumfaciens in the periwinkle in the presence of X. fastidiosa. X. fastidiosa induced stunting and reduced the number of flowers produced by the periwinkle. When C. flaccumfaciens was inoculated together with X. fastidiosa, no stunting was observed. The number of flowers produced by our doubly- inoculated plants was an intermediate between the number produced by the plants inoculated with either of the bacteria separately. Our data indicate that C. flaccumfaciens interacted with X. fastidiosa in C. roseus, and reduced the severity of the disease symptoms induced by X. fastidiosa. Periwinkle is considered to be an excellent experimental system by which the interaction of C. flaccumfaciens and other endophytic bacteria with X. fastidiosa can be

Transcriptome Analysis of the Phytobacterium Xylella fastidiosa Growing under Xylem-Based Chemical Conditions

PubMed Central

Ciraulo, Maristela Boaceff; Santos, Daiene Souza; Rodrigues, Ana Claudia de Freitas Oliveira; de Oliveira, Marcus Vinícius; Rodrigues, Tiago; de Oliveira, Regina Costa; Nunes, Luiz R.

2010-01-01

Xylella fastidiosa is a xylem-limited bacterium responsible for important plant diseases, like citrus-variegated chlorosis (CVC) and grapevine Pierce's disease (PD). Interestingly, in vitro growth of X. fastidiosa in chemically defined media that resemble xylem fluid has been achieved, allowing studies of metabolic processes used by xylem-dwelling bacteria to thrive in such nutrient-poor conditions. Thus, we performed microarray hybridizations to compare transcriptomes of X. fastidiosa cells grown in 3G10-R, a medium that resembles grape sap, and in Periwinkle Wilt (PW), the complex medium traditionally used to cultivate X. fastidiosa. We identified 299 transcripts modulated in response to growth in these media. Some 3G10R-overexpressed genes have been shown to be upregulated in cells directly isolated from infected plants and may be involved in plant colonization, virulence and environmental competition. In contrast, cells cultivated in PW show a metabolic switch associated with increased aerobic respiration and enhanced bacterial growth rates. PMID:20625415

Transcriptome analysis of the phytobacterium Xylella fastidiosa growing under xylem-based chemical conditions.

PubMed

Ciraulo, Maristela Boaceff; Santos, Daiene Souza; Rodrigues, Ana Claudia de Freitas Oliveira; de Oliveira, Marcus Vinícius; Rodrigues, Tiago; de Oliveira, Regina Costa; Nunes, Luiz R

2010-01-01

Xylella fastidiosa is a xylem-limited bacterium responsible for important plant diseases, like citrus-variegated chlorosis (CVC) and grapevine Pierce's disease (PD). Interestingly, in vitro growth of X. fastidiosa in chemically defined media that resemble xylem fluid has been achieved, allowing studies of metabolic processes used by xylem-dwelling bacteria to thrive in such nutrient-poor conditions. Thus, we performed microarray hybridizations to compare transcriptomes of X. fastidiosa cells grown in 3G10-R, a medium that resembles grape sap, and in Periwinkle Wilt (PW), the complex medium traditionally used to cultivate X. fastidiosa. We identified 299 transcripts modulated in response to growth in these media. Some 3G10R-overexpressed genes have been shown to be upregulated in cells directly isolated from infected plants and may be involved in plant colonization, virulence and environmental competition. In contrast, cells cultivated in PW show a metabolic switch associated with increased aerobic respiration and enhanced bacterial growth rates.

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil.

PubMed

Qin, X; Miranda, V S; Machado, M A; Lemos, E G; Hartung, J S

2001-06-01

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

Paradigms: examples from the bacterium Xylella fastidiosa.

PubMed

Purcell, Alexander

2013-01-01

The history of advances in research on Xylella fastidiosa provides excellent examples of how paradigms both advance and limit our scientific understanding of plant pathogens and the plant diseases they cause. I describe this from a personal perspective, having been directly involved with many persons who made paradigm-changing discoveries, beginning with the discovery that a bacterium, not a virus, causes Pierce's disease of grape and other plant diseases in numerous plant species, including important crop and forest species.

Xylella fastidiosa CoDiRO strain associated with the olive quick decline syndrome in southern Italy belongs to a clonal complex of the subspecies pauca that evolved in Central America.

PubMed

Marcelletti, Simone; Scortichini, Marco

2016-12-01

Xylella fastidiosa, a xylem-limited bacterium transmitted by xylem-fluid-feeding Hemiptera insects, causes economic losses of both woody and herbaceous plant species. A Xyl. fastidiosa subsp. pauca strain, namely CoDiRO, was recently found to be associated with the 'olive quick decline syndrome' in southern Italy (i.e. Apulia region). Recently, some Xyl. fastidiosa strains intercepted in France from Coffea spp. plant cuttings imported from Central and South America were characterized. The introduction of infected plant material from Central America in Apulia was also postulated even though an ad hoc study to confirm this hypothesis is lacking. In the present study, we assessed the complete and draft genome of 27 Xyl. fastidiosa strains. Through a genome-wide approach, we confirmed the occurrence of three subspecies within Xyl. fastidiosa, namely fastidiosa, multiplex and pauca, and demonstrated the occurrence of a genetic clonal complex of four Xyl. fastidiosa strains belonging to subspecies pauca which evolved in Central America. The CoDiRO strain displayed 13 SNPs when compared with a strain isolated in Costa Rica from Coffea sp. and 32 SNPs when compared with two strains obtained from Nerium oleander in Costa Rica. These results support the close relationships of the two strains. The four strains in the clonal complex contain prophage-like genes in their genomes. This study strongly supports the possibility of the introduction of Xyl. fastidiosa in southern Italy via coffee plants grown in Central America. The data also stress how the current global circulation of agricultural commodities potentially threatens the agrosystems worldwide.

Initial genetic analysis of Xylella fastidiosa in Texas.

PubMed

Morano, Lisa D; Bextine, Blake R; Garcia, Dennis A; Maddox, Shermel V; Gunawan, Stanley; Vitovsky, Natalie J; Black, Mark C

2008-04-01

Xylella fastidiosa is the causative agent of Pierce's Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.

Characterization of Xylella fastidiosa pear leaf scorch strain in Taiwan through whole genome sequence analyses

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a Gram-negative bacterium causing diseases in many economically important crops mostly in the Americas but also in Asia and Europe. A strain of X. fastidiosa was found to cause pear leaf scorch (PLS) disease in Taiwan in 1992. Because of nutritional fastidiousness, characteriza...

Evaluation of Arabidopsis thaliana as a model host for Xylella fastidiosa.

PubMed

Rogers, Elizabeth E

2012-06-01

The bacterium Xylella fastidiosa causes a number of plant diseases of significant economic impact. To date, progress determining mechanisms of host-plant susceptibility, tolerance, or resistance has been slow, due in large part to the long generation time and limited available genetic resources for grape, almond, and other known hosts of X. fastidiosa. To overcome many of these limitations, Arabidopsis thaliana has been evaluated as a host for X. fastidiosa. A pin-prick inoculation method has been developed to infect Arabidopsis with X. fastidiosa. Following infection, X. fastidiosa multiplies and can be detected by microscopy, polymerase chain reaction, and isolation. The ecotypes Van-0, LL-0, and Tsu-1 all allow more growth of strain X. fastidiosa Temecula than the reference ecotype Col-0. Affymetrix ATH1 microarray analysis of inoculated vs. noninoculated Tsu-1 reveals gene expression changes that differ greatly from changes seen after infection with apoplast-colonizing bacteria such as Psuedomonas syringae pvs. tomato or syringae. Many genes responsive to oxidative stress are differentially regulated, while classic pathogenesis-related genes are not induced by X. fastidiosa infection.

RNA metabolism in Xylella fastidiosa during cold adaptation and survival responses

USDA-ARS?s Scientific Manuscript database

Fastidious plant pathogen Xylella fastidiosa has a reduced ability to adapt to cold temperatures, limiting persistence in perennial hosts, such as grapevine, growing in colder regions. RNA metabolism is an essential part of bacterial response to low temperature, including inducible expression of RNA...

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins.

PubMed

Zaini, Paulo A; Fogaça, Andréa C; Lupo, Fernanda G N; Nakaya, Helder I; Vêncio, Ricardo Z N; da Silva, Aline M

2008-04-01

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2'-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 microM ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X. fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.

In vitro Determination of Extracellular Proteins from Xylella fastidiosa.

PubMed

Mendes, Juliano S; Santiago, André S; Toledo, Marcelo A S; Horta, Maria A C; de Souza, Alessandra A; Tasic, Ljubica; de Souza, Anete P

2016-01-01

The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa . Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa . Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3-30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components.

In vitro Determination of Extracellular Proteins from Xylella fastidiosa

PubMed Central

Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

2016-01-01

The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

PubMed

Roper, M Caroline; Greve, L Carl; Warren, Jeremy G; Labavitch, John M; Kirkpatrick, Bruce C

2007-04-01

Xylella fastidiosa is the causal agent of Pierce's disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

Functional characterization of the role of rpfA in Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa coordinates virulence in grapevines via quorum sensing signal molecules that are regulated and synthesized by the rpf gene cluster (regulation of pathogenicity factors). rpfA encodes aconitate hydratase and could play a regulator role involved in virulence. To elucidate the role o...

Xylella fastidiosa infection of grapevines affects host secondary metabolite and defense-related protein levels within xylem

USDA-ARS?s Scientific Manuscript database

Pierce’s disease of grapevine is a serious threat to grape production and is caused by the xylem-dwelling bacterial pathogen Xylella fastidiosa. Microscopy studies have documented morphological changes to grapevine xylem due to infection by X. fastidiosa. Comparatively, less is known about the bi...

Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay.

PubMed

Oliveira, Antonio C; Vallim, Marcelo A; Semighini, Camile P; Araújo, Welington L; Goldman, Gustavo H; Machado, Marcos A

2002-10-01

ABSTRACT Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease of sweet orange cultivars in Brazil. Polymerase chain reaction (PCR)-based assays constitute the principal diagnostic method for detection of these bacteria. In this work, we established a real-time quantitative PCR (QPCR) assay to quantify X. fastidiosa in naturally and artificially infected citrus. The X. fastidiosa cell number detected in the leaves increased according to the age of the leaf, and bacteria were not detected in the upper midrib section in young leaves, indicating temporal and spatial distribution patterns of bacteria, respectively. In addition, the X. fastidiosa cell number quantified in leaves of 'Pera' orange and 'Murcott' tangor reflected the susceptible and resistant status of these citrus cultivars. None of the 12 endophytic citrus bacteria or the four strains of X. fastidiosa nonpathogenic to citrus that were tested showed an increase in the fluorescence signal during QPCR. In contrast, all 10 CVC-causing strains exhibited an increase in fluorescence signal, thus indicating the specificity of this QPCR assay. Our QPCR provides a powerful tool for studies of different aspects of the Xylella-citrus interactions, and can be incorporated into breeding programs in order to select CVC-resistant plants more quickly.

Effects of rootstock on Xylella fastidiosa infection and grapevine sap phenolics

USDA-ARS?s Scientific Manuscript database

Pierce’s disease, caused by the bacterium Xylella fastidiosa, poses a threat to grape production in the United States and warm climates elsewhere. There are numerous grapevine rootstocks available that may impart increased vigor or tolerance to soil-borne pests. However, little is known about the po...

Multiplexed lateral flow microarray assay for detection of citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis pv citri

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cary,; Bruce, R; Stubben, Christopher J

The invention provides highly sensitive and specific assays for the major citrus pathogens Xylella fastidiosa and Xanthomonas axonopodis, including a field deployable multiplexed assay capable of rapidly assaying for both pathogens simultaneously. The assays are directed at particular gene targets derived from pathogenic strains that specifically cause the major citrus diseases of citrus variegated chlorosis (Xylella fastidiosa 9a5c) and citrus canker (Xanthomonas axonopodis pv citri). The citrus pathogen assays of the invention offer femtomole sensitivity, excellent linear dynamic range, and rapid and specific detection.

Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains.

PubMed

Brady, Jeff A; Faske, Jennifer B; Ator, Rebecca A; Castañeda-Gill, Jessica M; Mitchell, Forrest L

2012-04-01

Epidemiological studies of Pierce's disease (PD) can be confounded by a lack of taxonomic detail on the bacterial causative agent, Xylella fastidiosa (Xf). PD in grape is caused by strains of Xylella fastidiosa subsp. fastidiosa, but is not caused by other subspecies of Xf that typically colonize plants other than grape. Detection assays using ELISA and qPCR are effective at detecting and quantifying Xf presence or absence, but offer no information on Xf subspecies or strain identity. Surveying insects or host plants for Xf by current ELISA or qPCR methods provides only presence/absence and quantity information for any and all Xf subspecies, potentially leading to false assessments of disease threat. This study uses a series of adjacent-hybridizing DNA melt analysis probes that are capable of efficiently discriminating Xf subspecies and strain relationships in rapid real-time PCR reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Xylella fastidiosa plasmid-encoded PemK toxin is an endoribonuclease.

USDA-ARS?s Scientific Manuscript database

Stable inheritance of pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK plasmid addiction system. PemK serves as a toxin inhibiting bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK toxin by direct binding. PemK toxin and PemI antitoxin were over-expre...

Xylella fastidiosa gene expression analysis by DNA microarrays.

PubMed

Travensolo, Regiane F; Carareto-Alves, Lucia M; Costa, Maria V C G; Lopes, Tiago J S; Carrilho, Emanuel; Lemos, Eliana G M

2009-04-01

Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM(2) and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants.

The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a Gram-negative, xylem-limited pathogenic bacterium that causes Pierce’s disease of grapevines. Type IV pili of X. fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon involving signal transduction pathways. To elucidate the role of pilG in twitching motil...

Characterization of the LysR-type transcriptional regulator YcjZ-like from Xylella fastidiosa overexpressed in Escherichia coli.

PubMed

Santiago, André S; Santos, Clelton A; Mendes, Juliano S; Toledo, Marcelo A S; Beloti, Lilian L; Souza, Alessandra A; Souza, Anete P

2015-09-01

The Xylella fastidiosa 9a5c strain is a xylem-limited phytopathogen that is the causal agent of citrus variegated chlorosis (CVC). This bacterium is able to form a biofilm and occlude the xylem vessels of susceptible plants, which leads to significant agricultural and economic losses. Biofilms are associated with bacterial pathogenicity because they are very resistant to antibiotics and other metal-based chemicals that are used in agriculture. The X. fastidiosa YcjZ-like (XfYcjZ-like) protein belongs to the LysR-type transcriptional regulator (LTTR) family and is involved in various cellular functions that range from quorum sensing to bacterial survival. In the present study, we report the cloning, expression and purification of XfYcjZ-like, which was overexpressed in Escherichia coli. The secondary folding of the recombinant and purified protein was assessed by circular dichroism, which revealed that XfYcjZ-like contains a typical α/β fold. An initial hydrodynamic characterization showed that XfYcjZ-like is a globular tetramer in solution. In addition, using a polyclonal antibody against XfYcjZ-like, we assessed the expression profile of this protein during the different developmental phases of X. fastidiosa in in vitro cultivated biofilm cells and demonstrated that XfYcjZ-like is upregulated in planktonic cells in response to a copper shock treatment. Finally, the ability of XfYcjZ-like to interact with its own predicted promoter was confirmed in vitro, which is a typical feature of LysR. Taken together, our findings indicated that the XfYcjZ-like protein is involved in both the organization of the architecture and the maturation of the bacterial biofilm and that it is responsive to oxidative stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Xylella fastidiosa Isolates from Both subsp. multiplex and fastidiosa Cause Disease on Southern Highbush Blueberry (Vaccinium sp.) Under Greenhouse Conditions.

PubMed

Oliver, J E; Cobine, P A; De La Fuente, L

2015-07-01

Xylella fastidiosa is a xylem-limited gram-negative plant pathogen that affects numerous crop species, including grape, citrus, peach, pecan, and almond. Recently, X. fastidiosa has also been found to be the cause of bacterial leaf scorch on blueberry in the southeastern United States. Thus far, all X. fastidiosa isolates obtained from infected blueberry have been classified as X. fastidiosa subsp. multiplex; however, X. fastidiosa subsp. fastidiosa isolates are also present in the southeastern United States and commonly cause Pierce's disease of grapevines. In this study, seven southeastern U.S. isolates of X. fastidiosa, including three X. fastidiosa subsp. fastidiosa isolates from grape, one X. fastidiosa subsp. fastidiosa isolate from elderberry, and three X. fastidiosa subsp. multiplex isolates from blueberry, were used to infect the southern highbush blueberry 'Rebel'. Following inoculation, all isolates colonized blueberry, and isolates from both X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa caused symptoms, including characteristic stem yellowing and leaf scorch symptoms as well as dieback of the stem tips. Two X. fastidiosa subsp. multiplex isolates from blueberry caused more severe symptoms than the other isolates examined, and infection with these two isolates also had a significant impact on host mineral nutrient content in sap and leaves. These findings have potential implications for understanding X. fastidiosa host adaptation and expansion and the development of emerging diseases caused by this bacterium.

Diversity of Xylella fastidiosa host suitability among siblings from a non-traditional almond X peach cross

USDA-ARS?s Scientific Manuscript database

Ten F2 clones from an initial hybridization of Prunus webbii X P. persica cv Harrow Blood were evaluated under greenhouse conditions for their reaction to Xylella fastidiosa subsp. fastidiosa strain M23 during two growing seasons. Clonal accessions used for the study were selected on the basis of ho...

Analyses of Xylella whole genome sequences and proposal of Xylella taiwanensis sp. nov.

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a Gram negative, xylem limited and nutritionally fastidious plant pathogenic bacterium that cause disease in many economically important plants. A single species, fastidiosa, with three subspecies (fastidiosa, multiplex, and pauca) have been described. Most Xylella strains were...

Effect of oxygen on the growth and biofilm formation of Xylella fastidiosa in liquid media.

PubMed

Shriner, Anthony D; Andersen, Peter C

2014-12-01

Xylella fastidiosa is a xylem-limited bacterial pathogen, and is the causative agent of Pierce's disease of grapevines and scorch diseases of many other plant species. The disease symptoms are putatively due to blocking of the transpiration stream by bacterial-induced biofilm formation and/or by the formation of plant-generated tylosis. Xylella fastidiosa has been classified as an obligate aerobe, which appears unusual given that dissolved O2 levels in the xylem during the growing season are often hypoxic (20-60 μmol L(-1)). We examined the growth and biofilm formation of three strains of X. fastidiosa under variable O2 conditions (21, 2.1, 0.21 and 0 % O2), in comparison to that of Pseudomonas syringae (obligate aerobe) and Erwinia carotovora (facultative anaerobe) under similar conditions. The growth of X. fastidiosa more closely resembled that of the facultative anaerobe, and not the obligate aerobe. Xanthomonas campestris, the closest genetic relative of X. fastidiosa, exhibited no growth in an N2 environment, whereas X. fastidiosa was capable of growing in an N2 environment in PW(+), CHARDS, and XDM2-PR media. The magnitude of growth and biofilm formation in the N2 (0 % O2) treatment was dependent on the specific medium. Additional studies involving the metabolism of X. fastidiosa in response to low O2 are warranted. Whether X. fastidiosa is classified as an obligate aerobe or a facultative anaerobe should be confirmed by gene activation and/or the quantification of the metabolic profiles under hypoxic conditions.

Comparative Genomics- Identifying similarities and differences across three leafhopper vectors of Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Leafhoppers are the second most important vectors of agricultural diseases, thus we examined the gene expression across three leafhopper leafhoppers, Homalodisca vitripennis, Graphocephala atropunctata, and Oncometopia nigricans, which are vectors of the plant-infecting bacterium, Xylella fastidiosa...

Absence of classical heat shock response in the citrus pathogen Xylella fastidiosa.

PubMed

Martins-de-Souza, Daniel; Martins, Daniel; Astua-Monge, Gustavo; Coletta-Filho, Helvécio Della; Winck, Flavia Vischi; Baldasso, Paulo Aparecido; de Oliveira, Bruno Menezes; Marangoni, Sérgio; Machado, Marcos Antônio; Novello, José Camillo; Smolka, Marcus Bustamante

2007-02-01

The fastidious bacterium Xylella fastidiosa is associated with important crop diseases worldwide. We have recently shown that X. fastidiosa is a peculiar organism having unusually low values of gene codon bias throughout its genome and, unexpectedly, in the group of the most abundant proteins. Here, we hypothesized that the lack of codon usage optimization in X. fastidiosa would incapacitate this organism to undergo quick and massive changes in protein expression as occurs in a classical stress response. Proteomic analysis of the response to heat stress in X. fastidiosa revealed that no changes in protein expression can be detected. Moreover, stress-inducible proteins identified in the closely related citrus pathogen Xanthomonas axonopodis pv citri were found to be constitutively expressed in X. fastidiosa. These proteins have extremely high codon bias values in the X. citri and other well-studied organisms, but low values in X. fastidiosa. Because biased codon usage is well known to correlate to the rate of protein synthesis, we speculate that the peculiar codon bias distribution in X. fastidiosa is related to the absence of a classical stress response, and, probably, alternative strategies for survival of X. fastidiosa under stressfull conditions.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa.

PubMed

Shi, Xiangyang; Lin, Hong

2016-04-08

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant XfΔpilG and its complementary strain XfΔpilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in XfΔpilG, XfΔpilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary XfΔpilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant XfΔpliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Chitin utilization by the insect-transmitted bacterium Xylella fastidiosa.

PubMed

Killiny, Nabil; Prado, Simone S; Almeida, Rodrigo P P

2010-09-01

Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa.

Expression of green fluorescent protein in Xylella fastidiosa is affected by passage through host plants.

PubMed

Qin, Xiaoting; Hartung, John S

2004-09-01

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierce's disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.

Fastidian gum: the Xylella fastidiosa exopolysaccharide possibly involved in bacterial pathogenicity.

PubMed

da Silva, F R; Vettore, A L; Kemper, E L; Leite, A; Arruda, P

2001-09-25

The Gram-negative bacterium Xylella fastidiosa was the first plant pathogen to be completely sequenced. This species causes several economically important plant diseases, including citrus variegated chlorosis (CVC). Analysis of the genomic sequence of X. fastidiosa revealed a 12 kb DNA fragment containing an operon closely related to the gum operon of Xanthomonas campestris. The presence of all genes involved in the synthesis of sugar precursors, existence of exopolysaccharide (EPS) production regulators in the genome, and the absence of three of the X. campestris gum genes suggested that X. fastidiosa is able to synthesize an EPS different from that of xanthan gum. This novel EPS probably consists of polymerized tetrasaccharide repeating units assembled by the sequential addition of glucose-1-phosphate, glucose, mannose and glucuronic acid on a polyprenol phosphate carrier.

The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa.

PubMed

Nunney, L; Elfekih, S; Stouthamer, R

2012-05-01

Microbial identification methods have evolved rapidly over the last few decades. One such method is multilocus sequence typing (MLST). MLST is a powerful tool for understanding the evolutionary dynamics of pathogens and to gain insight into their genetic diversity. We illustrate the importance of accurate typing by reporting on three problems that have arisen in the study of a single bacterial species, the plant pathogen Xylella fastidiosa. Two of these were particularly serious since they concerned contamination of important research material that has had detrimental consequences for Xylella research: the contamination of DNA used in the sequencing of an X. fastidiosa genome (Ann-1) with DNA from another X. fastidiosa strain, and the unrecognized mislabeling of a strain (Temecula1) distributed from a culture collection (ATCC). We advocate the routine use of MLST to define strains maintained in culture collections and emphasize the importance of confirming the purity of DNA submitted for sequencing. We also present a third example that illustrates the value of MLST in guiding the choice of taxonomic types. Beyond these situations, there is a strong case for MLST whenever an isolate is used experimentally, especially where genotypic differences are suspected to influence the outcome.

Genetic analysis of a novel Xylella fastidiosa subspecies found in the southwestern United States.

PubMed

Randall, Jennifer J; Goldberg, Natalie P; Kemp, John D; Radionenko, Maxim; French, Jason M; Olsen, Mary W; Hanson, Stephen F

2009-09-01

Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.

Arabidopsis Thaliana Ecotypes With Differential Susceptibility To The Bacterial Pathogen Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Pierce’s disease of grapes and almond leaf scorch are devastating diseases caused by the bacterium Xylella fastidiosa (Xf). To date, progress in determining the mechanisms of host plant susceptibility, tolerance or resistance has been slow, due in large part to the long generation time and limited a...

Grapevines undergo varying shifts in secondary metabolic profiles when infected with Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Pierce’s disease (PD) is a devastating disease of grapevine caused by the bacterial pathogen Xylella fastidiosa (Xf). Key to the development and optimization of PD-tolerant grape cultivars is improved understanding about how grapevines defend themselves against Xf. This study complements histologica...

The DinJ/RelE toxin-antitoxin system suppresses virulence in Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa, the causal agent of a number agriculturally important plant diseases, encodes multiple toxin-antitoxin (TA) systems. TA modules consist of a toxin protein co-expressed with a specific antitoxin, and are often acquired through horizontal gene transfer. Antitoxin molecules (RNA or ...

Microarray analyses of Xylella fastidiosa provide evidence of coordinated transcription control of laterally transferred elements.

PubMed

Nunes, Luiz R; Rosato, Yoko B; Muto, Nair H; Yanai, Giane M; da Silva, Vivian S; Leite, Daniela B; Gonçalves, Edmilson R; de Souza, Alessandra A; Coletta-Filho, Helvécio D; Machado, Marcos A; Lopes, Silvio A; de Oliveira, Regina Costa

2003-04-01

Genetically distinct strains of the plant bacterium Xylella fastidiosa (Xf) are responsible for a variety of plant diseases, accounting for severe economic damage throughout the world. Using as a reference the genome of Xf 9a5c strain, associated with citrus variegated chlorosis (CVC), we developed a microarray-based comparison involving 12 Xf isolates, providing a thorough assessment of the variation in genomic composition across the group. Our results demonstrate that Xf displays one of the largest flexible gene pools characterized to date, with several horizontally acquired elements, such as prophages, plasmids, and genomic islands (GIs), which contribute up to 18% of the final genome. Transcriptome analysis of bacteria grown under different conditions shows that most of these elements are transcriptionally active, and their expression can be influenced in a coordinated manner by environmental stimuli. Finally, evaluation of the genetic composition of these laterally transferred elements identified differences that may help to explain the adaptability of Xf strains to infect such a wide range of plant species.

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes.

PubMed

Guilhabert, M R; Hoffman, L M; Mills, D A; Kirkpatrick, B C

2001-06-01

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

PubMed

Simpson, A J; Reinach, F C; Arruda, P; Abreu, F A; Acencio, M; Alvarenga, R; Alves, L M; Araya, J E; Baia, G S; Baptista, C S; Barros, M H; Bonaccorsi, E D; Bordin, S; Bové, J M; Briones, M R; Bueno, M R; Camargo, A A; Camargo, L E; Carraro, D M; Carrer, H; Colauto, N B; Colombo, C; Costa, F F; Costa, M C; Costa-Neto, C M; Coutinho, L L; Cristofani, M; Dias-Neto, E; Docena, C; El-Dorry, H; Facincani, A P; Ferreira, A J; Ferreira, V C; Ferro, J A; Fraga, J S; França, S C; Franco, M C; Frohme, M; Furlan, L R; Garnier, M; Goldman, G H; Goldman, M H; Gomes, S L; Gruber, A; Ho, P L; Hoheisel, J D; Junqueira, M L; Kemper, E L; Kitajima, J P; Krieger, J E; Kuramae, E E; Laigret, F; Lambais, M R; Leite, L C; Lemos, E G; Lemos, M V; Lopes, S A; Lopes, C R; Machado, J A; Machado, M A; Madeira, A M; Madeira, H M; Marino, C L; Marques, M V; Martins, E A; Martins, E M; Matsukuma, A Y; Menck, C F; Miracca, E C; Miyaki, C Y; Monteriro-Vitorello, C B; Moon, D H; Nagai, M A; Nascimento, A L; Netto, L E; Nhani, A; Nobrega, F G; Nunes, L R; Oliveira, M A; de Oliveira, M C; de Oliveira, R C; Palmieri, D A; Paris, A; Peixoto, B R; Pereira, G A; Pereira, H A; Pesquero, J B; Quaggio, R B; Roberto, P G; Rodrigues, V; de M Rosa, A J; de Rosa, V E; de Sá, R G; Santelli, R V; Sawasaki, H E; da Silva, A C; da Silva, A M; da Silva, F R; da Silva, W A; da Silveira, J F; Silvestri, M L; Siqueira, W J; de Souza, A A; de Souza, A P; Terenzi, M F; Truffi, D; Tsai, S M; Tsuhako, M H; Vallada, H; Van Sluys, M A; Verjovski-Almeida, S; Vettore, A L; Zago, M A; Zatz, M; Meidanis, J; Setubal, J C

2000-07-13

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Characterization of novel virulent broad-host-range phages of Xylella fastidiosa and Xanthomonas.

PubMed

Ahern, Stephen J; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry; Gonzalez, Carlos F

2014-01-01

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ~4 × 10(-12) ml cell(-1) min(-1) for X. fastidiosa strain Temecula 1 and ~5 × 10(-10) to 7 × 10(-10) ml cell(-1) min(-1) for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.

Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

PubMed Central

Ahern, Stephen J.; Das, Mayukh; Bhowmick, Tushar Suvra; Young, Ry

2014-01-01

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce's disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ∼4 × 10−12 ml cell−1 min−1 for X. fastidiosa strain Temecula 1 and ∼5 × 10−10 to 7 × 10−10 ml cell−1 min−1 for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa. PMID:24214944

Glassy-winged sharpshooter can use a mechanical mechanism to inoculate Xylella fastidiosa into grapevines

USDA-ARS?s Scientific Manuscript database

Xylem-feeding leafhoppers such as the glassy-winged sharpshooter, Homalodisca vitripennis (Cicadellidae: Cicadellinae), are thought to inoculate the bacterium Xylella fastidiosa (Xf) from colonies bound to cuticle of the sharpshooter’s functional foregut (precibarium and cibarium). The mechanism of ...

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis.

PubMed

Lacava, P T; Araújo, W L; Marcon, J; Maccheroni, W; Azevedo, J L

2004-01-01

To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.

Evaluation of pathogenicity and insect transmission of Xylella fastidiosa strains to olive plants

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa (Xf) is a xylem-limited bacterium that causes disease in a number of economically important crops in California and worldwide. Newly observed scorching symptoms in olive trees may be due to Xf infection. If true, “olive leaf scorch disease” (OLSD) would represent a new threat to...

Whole genome sequence analyses of Xylella fastidiosa PD strains from different geographical regions

USDA-ARS?s Scientific Manuscript database

Genome sequences were determined for two Pierce’s disease (PD) causing Xylella fastidiosa (Xf) strains, one from Florida and one from Taiwan. The Florida strain was ATCC 35879, the type of strain used as a standard reference for related taxonomy research. By contrast, the Taiwan strain used was only...

Transmission of Xylella fastidiosa to Grapevine by the Meadow Spittlebug.

PubMed

Cornara, D; Sicard, A; Zeilinger, A R; Porcelli, F; Purcell, A H; Almeida, R P P

2016-11-01

There is little information available on Xylella fastidiosa transmission by spittlebugs (Hemiptera, Cercopoidea). This group of insect vectors may be of epidemiological relevance in certain diseases, so it is important to better understand the basic parameters of X. fastidiosa transmission by spittlebugs. We used grapevines as a host plant and the aphrophorid Philaenus spumarius as a vector to estimate the effect of plant access time on X. fastidiosa transmission to plants; in addition, bacterial population estimates in the heads of vectors were determined and correlated with plant infection status. Results show that transmission efficiency of X. fastidiosa by P. spumarius increased with plant access time, similarly to insect vectors in another family (Hemiptera, Cicadellidae). Furthermore, a positive correlation between pathogen populations in P. spumarius and transmission to plants was observed. Bacterial populations in insects were one to two orders of magnitude lower than those observed in leafhopper vectors, and population size peaked within 3 days of plant access period. These results suggest that P. spumarius has either a limited number of sites in the foregut that may be colonized, or that fluid dynamics in the mouthparts of these insects is different from that in leafhoppers. Altogether our results indicate that X. fastidiosa transmission by spittlebugs is similar to that by leafhoppers. In addition, the relationship between cell numbers in vectors and plant infection may have under-appreciated consequences to pathogen spread.

Draft genome sequence of Xylella fastidiosa pear leaf scorch strain in Taiwan

USDA-ARS?s Scientific Manuscript database

The draft genome sequence of Xylella fastidiosa pear leaf scorch strain (PLS229) isolated from pear cultivar Hengshan (Pyrus pyrifolia) in Taiwan is reported. The bacterium has a genome size of 2,733,013 bp with a G+C content of 53.1%. The PLS229 strain genome was annotated to have 3,259 open readin...

Distribution of Xylella fastidiosa in Sycamore associated with low temperature and host resistance

Treesearch

T.S.M. Henneberger; K.L. Stevenson; C.J. Chang

2004-01-01

Experiments were conducted in the field and laboratory to determine effects of low temperatures 4% on Xylella fastidiosa populations in American sycamore. Roots and shoots from naturally infected trees at two locations were collected monthly. Sap extracted from the samples was tested by enzyme-linked immunosorbent assay for presence of X...

The Complex Biogeography of the Plant Pathogen Xylella fastidiosa: Genetic Evidence of Introductions and Subspecific Introgression in Central America

PubMed Central

Nunney, Leonard; Ortiz, Beatriz; Russell, Stephanie A.; Ruiz Sánchez, Rebeca; Stouthamer, Richard

2014-01-01

The bacterium Xylella fastidiosa is a plant pathogen with a history of economically damaging introductions of subspecies to regions where its other subspecies are native. Genetic evidence is presented demonstrating the introduction of two new taxa into Central America and their introgression into the native subspecies, X. fastidiosa subsp. fastidiosa. The data are from 10 genetic outliers detected by multilocus sequence typing (MLST) of isolates from Costa Rica. Six (five from oleander, one from coffee) defined a new sequence type (ST53) that carried alleles at six of the eight loci sequenced (five of the seven MLST loci) diagnostic of the South American subspecies Xylella fastidiosa subsp. pauca which causes two economically damaging plant diseases, citrus variegated chlorosis and coffee leaf scorch. The two remaining loci of ST53 carried alleles from what appears to be a new South American form of X. fastidiosa. Four isolates, classified as X. fastidiosa subsp. fastidiosa, showed a low level of introgression of non-native DNA. One grapevine isolate showed introgression of an allele from X. fastidiosa subsp. pauca while the other three (from citrus and coffee) showed introgression of an allele with similar ancestry to the alleles of unknown origin in ST53. The presence of X. fastidiosa subsp. pauca in Central America is troubling given its disease potential, and establishes another route for the introduction of this economically damaging subspecies into the US or elsewhere, a threat potentially compounded by the presence of a previously unknown form of X. fastidiosa. PMID:25379725

The complex biogeography of the plant pathogen Xylella fastidiosa: genetic evidence of introductions and Subspecific introgression in Central America.

PubMed

Nunney, Leonard; Ortiz, Beatriz; Russell, Stephanie A; Ruiz Sánchez, Rebeca; Stouthamer, Richard

2014-01-01

The bacterium Xylella fastidiosa is a plant pathogen with a history of economically damaging introductions of subspecies to regions where its other subspecies are native. Genetic evidence is presented demonstrating the introduction of two new taxa into Central America and their introgression into the native subspecies, X. fastidiosa subsp. fastidiosa. The data are from 10 genetic outliers detected by multilocus sequence typing (MLST) of isolates from Costa Rica. Six (five from oleander, one from coffee) defined a new sequence type (ST53) that carried alleles at six of the eight loci sequenced (five of the seven MLST loci) diagnostic of the South American subspecies Xylella fastidiosa subsp. pauca which causes two economically damaging plant diseases, citrus variegated chlorosis and coffee leaf scorch. The two remaining loci of ST53 carried alleles from what appears to be a new South American form of X. fastidiosa. Four isolates, classified as X. fastidiosa subsp. fastidiosa, showed a low level of introgression of non-native DNA. One grapevine isolate showed introgression of an allele from X. fastidiosa subsp. pauca while the other three (from citrus and coffee) showed introgression of an allele with similar ancestry to the alleles of unknown origin in ST53. The presence of X. fastidiosa subsp. pauca in Central America is troubling given its disease potential, and establishes another route for the introduction of this economically damaging subspecies into the US or elsewhere, a threat potentially compounded by the presence of a previously unknown form of X. fastidiosa.

Evidence that explains absence of a latent period for Xylella fastidiosa in its sharpshooter vectors

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar), and other sharpshooter (Cicadelline) leafhoppers transmit Xylella fastidiosa (Xf), the causative agent of Pierce’s disease of grapevine and other scorch diseases. Past research has supported that vectors have virtually no late...

Whole genome sequencing and analyses of Xylella fastidiosa subsp. fastidiosa strain GV156 causing Pierce’s disease of grapevine in Taiwan

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a nutritionally fastidious Gram-negative bacterium causing Pierce’s disease (PD) of grapevines. PD was first reported in Anaheim, California in 1892 and is currently endemic in California and the southeastern U.S. PD also was found outside the U.S. but is limited to the America...

The role of Xylella fastidiosa cold shock proteins in Pierce’s disease of grapes

USDA-ARS?s Scientific Manuscript database

Pierce’s disease of grapevine, caused by the bacterial pathogen Xylella fastidiosa (Xf) is limited to warmer climates, and plant infection can be eliminated by cold winter conditions. Milder winters can increase the likelihood of pathogen persistence from one growing season to the next. Cold adaptat...

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination.

PubMed

Jacques, Marie-Agnès; Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

2015-12-28

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination

PubMed Central

Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

2015-01-01

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. PMID:26712553

Potential complications when developing gene deletion clones in Xylella fastidiosa.

PubMed

Johnson, Kameka L; Cursino, Luciana; Athinuwat, Dusit; Burr, Thomas J; Mowery, Patricia

2015-04-16

The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers. Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations. We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present. We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

Genetic differences between two strains of Xylella fastidiosa revealed by suppression subtractive hybridization.

PubMed

Harakava, Ricardo; Gabriel, Dean W

2003-02-01

Suppression subtractive hybridization was used to rapidly identify 18 gene differences between a citrus variegated chlorosis (CVC) strain and a Pierce's disease of grape (PD) strain of Xylella fastidiosa. The results were validated as being highly representative of actual differences by comparison of the completely sequenced genome of a CVC strain with that of a PD strain.

Genetic discovery in Xylella fastidiosa through sequence analysis of selected randomly amplified polymorphic DNAs.

PubMed

Chen, Jianchi; Civerolo, Edwin L; Jarret, Robert L; Van Sluys, Marie-Anne; de Oliveira, Mariana C

2005-02-01

Xylella fastidiosa causes many important plant diseases including Pierce's disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the "phage" is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.

Identification of a low copy number plasmid in Xylella fastidiosa Strain Stag’s Leap

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa (Xf) causes Pierce’s Disease (PD) in grapevine. The Stag’s Leap strain is known for its high virulence level and is a model for PD research. Research on Xf has been difficult due to its nutritional fastidiousness. One difficult research issue is the low copy number plasmid. Plasmi...

Detection and typing of Xylella fastidiosa from glassy-winged sharpshooter for Pierce’s disease epidemiology

USDA-ARS?s Scientific Manuscript database

Epidemiology of Pierce’s disease of grape, caused by the bacterial pathogen Xylella fastidiosa (Xf), is largely dependent on populations of insect vectors such as the invasive glassy-winged sharpshooter (GWSS) (Homalodisca vitripennis). In the grape-growing regions of the southern San Joaquin Valley...

Toxin-antitoxin systems mqsR/ygiT and dinJ/RelE of Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

The plant pathogen Xylella fastidiosa (Xf) encodes multiple toxin-antitoxin (TA) system homologues, including relE/dinJ and mqsR/ygiT. Phylogenetic analyses indicate these two Xf TA systems have distinct evolutionary histories. Genomic comparisons among Xf subspecies/strains reveal TA systems are ...

Cell-cell signaling controls Xylella fastidiosa interactions with both insects and plants

PubMed Central

Newman, Karyn L.; Almeida, Rodrigo P. P.; Purcell, Alexander H.; Lindow, Steven E.

2004-01-01

Xylella fastidiosa, which causes Pierce's disease of grapevine and other important plant diseases, is a xylem-limited bacterium that depends on insect vectors for transmission. Although many studies have addressed disease symptom development and transmission of the pathogen by vectors, little is known about the bacterial mechanisms driving these processes. Recently available X. fastidiosa genomic sequences and molecular tools have provided new routes for investigation. Here, we show that a diffusible signal molecule is required for biofilm formation in the vector and for vector transmission to plants. We constructed strains of X. fastidiosa mutated in the rpfF gene and determined that they are unable to produce the signal activity. In addition, rpfF mutants are more virulent than the wild type when mechanically inoculated into plants. This signal therefore directs interaction of X. fastidiosa with both its insect vector and plant host. Interestingly, rpfF mutants can still form in planta biofilms, which differ architecturally from biofilms in insects, suggesting that biofilm architecture, rather than a passive response to the environment, is actively determined by X. fastidiosa gene expression. This article reports a cell-cell signaling requirement for vector transmission. Identification of the genes regulated by rpfF should elucidate bacterial factors involved in transmission and biofilm formation in the insect. PMID:14755059

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa.

PubMed

Cursino, Luciana; Galvani, Cheryl D; Athinuwat, Dusit; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2011-10-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

Habitat Effects on Population Density and Movement of Insect Vectors of Xylella fastidiosa in California, USA

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a xylem-limited bacterium that causes disease in grapevines, almonds, citrus, pear, alfalfa, and many other economically important plants. In California, USA, the bacteria are transmitted by several species of leafhoppers including the cicadellids Draeculacephala minerva Ball a...

Grapevine phenolics in xylem sap and tissues are significantly altered during infection by Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Pierce’s disease of grapevine (PD), caused by the bacterial pathogen Xylella fastidiosa (X.f.), remains a serious problem for grape production in California and elsewhere. This research examined induction of phenolic compounds in grapevines (cv. Thompson Seedless) infected with X.f. over a six month...

Twitching motility and biofilm formation are associated with tonB1 in Xylella fastidiosa.

PubMed

Cursino, Luciana; Li, Yaxin; Zaini, Paulo A; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J

2009-10-01

A mutation in the Xylella fastidiosa tonB1 gene resulted in loss of twitching motility and in significantly less biofilm formation as compared with a wild type. The altered motility and biofilm phenotypes were restored by complementation with a functional copy of the gene. The mutation affected virulence as measured by Pierce's disease symptoms on grapevines. The role of TonB1 in twitching and biofilm formation appears to be independent of the characteristic iron-uptake function of this protein. This is the first report demonstrating a functional role for a tonB homolog in X. fastidiosa.

Expression of Xylella fastidiosa RpfF in citrus disrupts signaling in Xanthomonas citri subsp. citri and thereby its virulence.

PubMed

Caserta, R; Picchi, S C; Takita, M A; Tomaz, J P; Pereira, W E L; Machado, M A; Ionescu, M; Lindow, S; De Souza, A A

2014-11-01

Xylella fastidiosa and Xanthomonas citri subsp. citri, that cause citrus variegated chlorosis (CVC) and citrus canker diseases, respectively, utilize diffusible signal factor (DSF) for quorum sensing. DSF, produced by RpfF, are similar fatty acids in both organisms, although a different set of genes is regulated by DSF in each species. Because of this similarity, Xylella fastidiosa DSF might be recognized and affect the biology of Xanthomonas citri. Therefore, transgenic Citrus sinensis and Carrizo citrange plants overexpressing the Xylella fastidiosa rpfF were inoculated with Xanthomonas citri and changes in symptoms of citrus canker were observed. X. citri biofilms formed only at wound sites on transgenic leaves and were thicker; however, bacteria were unable to break through the tissue and form pustules elsewhere. Although abundant growth of X. citri occurred at wound sites on inoculated transgenic leaves, little growth was observed on unwounded tissue. Genes in the DFS-responsive core in X. citri were downregulated in bacteria isolated from transgenic leaves. DSF-dependent expression of engA was suppressed in cells exposed to xylem sap from transgenic plants. Thus, altered symptom development appears to be due to reduced expression of virulence genes because of the presence of antagonists of DSF signaling in X. citri in rpfF-expressing plants.

Xylella fastidiosa infection of grapevines affects xylem levels of phenolic compounds and pathogenesis-related proteins

USDA-ARS?s Scientific Manuscript database

Pierce’s disease (PD), caused by the xylem-dwelling pathogen Xylella fastidiosa (X.f.), is a serious threat to grape production. The effects of X.f. infection six months post-inoculation on defense-associated proteins and phenolic compounds found in xylem sap and tissue were evaluated. Defense-assoc...

Structural and Biochemical Characterization of Xylella fastidiosa DsbA Family Members: New insightsinto the Enzyme-Substrate Interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rinaldi, F.; Meza, A; Gulmarges, B

2009-01-01

Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determinationmore » of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.« less

A suitable Xylella fastidiosa CVC strain for post-genome studies.

PubMed

Teixeira, Diva do Carmo; Rocha, Sanvai Regina Prado; de Santos, Mateus Almeida; Mariano, Anelise Galdino; Li, Wen Bin; Monteiro, Patricia Brant

2004-12-01

The genome sequence of the pathogen Xylella fastidiosa Citrus Variegated Chlorosis (CVC) strain 9a5c has revealed many genes related to pathogenicity mechanisms and virulence determinants. However, strain 9a5c is resistant to genetic transformation, impairing mutant production for the analysis of pathogenicity mechanisms and virulence determinants of this fastidious phytopathogen. By screening different strains, we found out that cloned strains J1a12, B111, and S11400, all isolated from citrus trees affected by CVC, are amenable to transformation, and J1a12 has been used as a model strain in a functional genomics program supported by FAPESP (São Paulo State Research Foundation). However, we have found that strain J1a12, unlike strains 9a5c and B111, was incapable of inducing CVC symptoms when inoculated in citrus plants. We have now determined that strain B111 is an appropriate candidate for post-genome studies of the CVC strain of X. fastidiosa.

Promiscuous Diffusible Signal Factor Production and Responsiveness of the Xylella fastidiosa Rpf System

PubMed Central

Ionescu, Michael; Yokota, Kenji; Antonova, Elena; Garcia, Angelica; Beaulieu, Ellen; Hayes, Terry; Iavarone, Anthony T.

2016-01-01

ABSTRACT Cell density-dependent regulation of gene expression in Xylella fastidiosa that is crucial to its switching between plant hosts and insect vectors is dependent on RpfF and its production of 2-enoic acids known as diffusible signal factor (DSF). We show that X. fastidiosa produces a particularly large variety of similar, relatively long-chain-length 2-enoic acids that are active in modulating gene expression. Both X. fastidiosa itself and a Pantoea agglomerans surrogate host harboring X. fastidiosa RpfF (XfRpfF) is capable of producing a variety of both saturated and unsaturated free fatty acids. However, only 2-cis unsaturated acids were found to be biologically active in X. fastidiosa. X. fastidiosa produces, and is particularly responsive to, a novel DSF species, 2-cis-hexadecanoic acid that we term XfDSF2. It is also responsive to other, even longer 2-enoic acids to which other taxa such as Xanthomonas campestris are unresponsive. The 2-enoic acids that are produced by X. fastidiosa are strongly affected by the cellular growth environment, with XfDSF2 not detected in culture media in which 2-tetradecenoic acid (XfDSF1) had previously been found. X. fastidiosa is responsive to much lower concentrations of XfDSF2 than XfDSF1. Apparently competitive interactions can occur between various saturated and unsaturated fatty acids that block the function of those agonistic 2-enoic fatty acids. By altering the particular 2-enoic acids produced and the relative balance of free enoic and saturated fatty acids, X. fastidiosa might modulate the extent of DSF-mediated quorum sensing. PMID:27435463

The Secreted Protease PrtA Controls Cell Growth, Biofilm Formation and Pathogenicity in Xylella fastidiosa.

PubMed

Gouran, Hossein; Gillespie, Hyrum; Nascimento, Rafael; Chakraborty, Sandeep; Zaini, Paulo A; Jacobson, Aaron; Phinney, Brett S; Dolan, David; Durbin-Johnson, Blythe P; Antonova, Elena S; Lindow, Steven E; Mellema, Matthew S; Goulart, Luiz R; Dandekar, Abhaya M

2016-08-05

Pierce's disease (PD) is a deadly disease of grapevines caused by the Gram-negative bacterium Xylella fastidiosa. Though disease symptoms were formerly attributed to bacteria blocking the plant xylem, this hypothesis is at best overly simplistic. Recently, we used a proteomic approach to characterize the secretome of X. fastidiosa, both in vitro and in planta, and identified LesA as one of the pathogenicity factors of X. fastidiosa in grapevines that leads to leaf scorching and chlorosis. Herein, we characterize another such factor encoded by PD0956, designated as an antivirulence secreted protease "PrtA" that displays a central role in controlling in vitro cell proliferation, length, motility, biofilm formation, and in planta virulence. The mutant in X. fastidiosa exhibited reduced cell length, hypermotility (and subsequent lack of biofilm formation) and hypervirulence in grapevines. These findings are supported by transcriptomic and proteomic analyses with corresponding plant infection data. Of particular interest, is the hypervirulent response in grapevines observed when X. fastidiosa is disrupted for production of PrtA, and that PD-model tobacco plants transformed to express PrtA exhibited decreased symptoms after infection by X. fastidiosa.

The Secreted Protease PrtA Controls Cell Growth, Biofilm Formation and Pathogenicity in Xylella fastidiosa

PubMed Central

Gouran, Hossein; Gillespie, Hyrum; Nascimento, Rafael; Chakraborty, Sandeep; Zaini, Paulo A.; Jacobson, Aaron; Phinney, Brett S.; Dolan, David; Durbin-Johnson, Blythe P.; Antonova, Elena S.; Lindow, Steven E.; Mellema, Matthew S.; Goulart, Luiz R.; Dandekar, Abhaya M.

2016-01-01

Pierce’s disease (PD) is a deadly disease of grapevines caused by the Gram-negative bacterium Xylella fastidiosa. Though disease symptoms were formerly attributed to bacteria blocking the plant xylem, this hypothesis is at best overly simplistic. Recently, we used a proteomic approach to characterize the secretome of X. fastidiosa, both in vitro and in planta, and identified LesA as one of the pathogenicity factors of X. fastidiosa in grapevines that leads to leaf scorching and chlorosis. Herein, we characterize another such factor encoded by PD0956, designated as an antivirulence secreted protease “PrtA” that displays a central role in controlling in vitro cell proliferation, length, motility, biofilm formation, and in planta virulence. The mutant in X. fastidiosa exhibited reduced cell length, hypermotility (and subsequent lack of biofilm formation) and hypervirulence in grapevines. These findings are supported by transcriptomic and proteomic analyses with corresponding plant infection data. Of particular interest, is the hypervirulent response in grapevines observed when X. fastidiosa is disrupted for production of PrtA, and that PD-model tobacco plants transformed to express PrtA exhibited decreased symptoms after infection by X. fastidiosa. PMID:27492542

Crystal structure of a small heat-shock protein from Xylella fastidiosa reveals a distinct high-order structure.

PubMed

Fonseca, Emanuella Maria Barreto; Scorsato, Valéria; Dos Santos, Marcelo Leite; Júnior, Atilio Tomazini; Tada, Susely Ferraz Siqueira; Dos Santos, Clelton Aparecido; de Toledo, Marcelo Augusto Szymanski; de Souza, Anete Pereira; Polikarpov, Igor; Aparicio, Ricardo

2017-04-01

Citrus variegated chlorosis is a disease that attacks economically important citrus plantations and is caused by the plant-pathogenic bacterium Xylella fastidiosa. In this work, the structure of a small heat-shock protein from X. fastidiosa (XfsHSP17.9) is reported. The high-order structures of small heat-shock proteins from other organisms are arranged in the forms of double-disc, hollow-sphere or spherical assemblies. Unexpectedly, the structure reported here reveals a high-order architecture forming a nearly square cavity.

Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by gacA.

PubMed

Shi, Xiang Yang; Dumenyo, C Korsi; Hernandez-Martinez, Rufina; Azad, Hamid; Cooksey, Donald A

2009-04-01

The xylem-limited, insect-transmitted bacterium Xylella fastidiosa causes Pierce's disease in grapes through cell aggregation and vascular clogging. GacA controls various physiological processes and pathogenicity factors in many gram-negative bacteria, including biofilm formation in Pseudomonas syringae pv. tomato DC3000. Cloned gacA of X. fastidiosa was found to restore the hypersensitive response and pathogenicity in gacA mutants of P. syringae pv. tomato DC3000 and Erwinia amylovora. A gacA mutant of X. fastidiosa (DAC1984) had significantly reduced abilities to adhere to a glass surface, form biofilm, and incite disease symptoms on grapevines, compared with the parent (A05). cDNA microarray analysis identified 7 genes that were positively regulated by GacA, including xadA and hsf, predicted to encode outer membrane adhesion proteins, and 20 negatively regulated genes, including gumC and an antibacterial polypeptide toxin gene, cvaC. These results suggest that GacA of X. fastidiosa regulates many factors, which contribute to attachment and biofilm formation, as well as some physiological processes that may enhance the adaptation and tolerance of X. fastidiosa to environmental stresses and the competition within the host xylem.

Multilocus sequence typing of Xylella fastidiosa causing Pierce's disease and oleander leaf scorch in the United States.

PubMed

Yuan, Xiaoli; Morano, Lisa; Bromley, Robin; Spring-Pearson, Senanu; Stouthamer, Richard; Nunney, Leonard

2010-06-01

Using a modified multilocus sequence typing (MLST) scheme for the bacterial plant pathogen Xylella fastidiosa based on the same seven housekeeping genes employed in a previously published MLST, we studied the genetic diversity of two subspecies, X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi, which cause Pierce's disease and oleander leaf scorch, respectively. Typing of 85 U.S. isolates (plus one from northern Mexico) of X. fastidiosa subsp. fastidiosa from 15 different plant hosts and 21 isolates of X. fastidiosa subsp. sandyi from 4 different hosts in California and Texas supported their subspecific status. Analysis using the MLST genes plus one cell-surface gene showed no significant genetic differentiation based on geography or host plant within either subspecies. Two cases of homologous recombination (with X. fastidiosa subsp. multiplex, the third U.S. subspecies) were detected in X. fastidiosa subsp. fastidiosa. Excluding recombination, MLST site polymorphism in X. fastidiosa subsp. fastidiosa (0.048%) and X. fastidiosa subsp. sandyi (0.000%) was substantially lower than in X. fastidiosa subsp. multiplex (0.240%), consistent with the hypothesis that X. fastidiosa subspp. fastidiosa and sandyi were introduced into the United States (probably just prior to 1880 and 1980, respectively). Using whole-genome analysis, we showed that MLST is more effective at genetic discrimination at the specific and subspecific level than other typing methods applied to X. fastidiosa. Moreover, MLST is the only technique effective in detecting recombination.

Expression of Xylella fastidiosa Fimbrial and Afimbrial Proteins during Biofilm Formation▿

PubMed Central

Caserta, R.; Takita, M. A.; Targon, M. L.; Rosselli-Murai, L. K.; de Souza, A. P.; Peroni, L.; Stach-Machado, D. R.; Andrade, A.; Labate, C. A.; Kitajima, E. W.; Machado, M. A.; de Souza, A. A.

2010-01-01

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants. PMID:20472735

VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

PubMed

Mendes, Juliano S; Santiago, André da S; Toledo, Marcelo A S; Rosselli-Murai, Luciana K; Favaro, Marianna T P; Santos, Clelton A; Horta, Maria Augusta C; Crucello, Aline; Beloti, Lilian L; Romero, Fabian; Tasic, Ljubica; de Souza, Alessandra A; de Souza, Anete P

2015-01-01

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

Structural and biochemical characterization of Xylella fastidiosa DsbA family members: new insights into the enzyme-substrate interaction.

PubMed

Rinaldi, Fábio C; Meza, Andréia N; Guimarães, Beatriz G

2009-04-21

Disulfide oxidoreductase DsbA catalyzes disulfide bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is among the most oxidizing of the thioredoxin-like proteins, and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family, and in one of them, the canonical motif CPHC is replaced by CPAC. Biochemical assays showed that both X. fastidiosa homologues have similar redox properties and the determination of the crystal structure of XfDsbA revealed substitutions in the active site of X. fastidiosa enzymes, which are proposed to compensate for the lack of the conserved histidine in XfDsbA2. In addition, electron density maps showed a ligand bound to the XfDsbA active site, allowing the characterization of the enzyme interaction with an 8-mer peptide. Finally, surface analysis of XfDsbA and XfDsbA2 suggests that X. fastidiosa enzymes may have different substrate specificities.

Calcium-Enhanced Twitching Motility in Xylella fastidiosa Is Linked to a Single PilY1 Homolog

PubMed Central

Cruz, Luisa F.; Parker, Jennifer K.; Cobine, Paul A.

2014-01-01

The plant-pathogenic bacterium Xylella fastidiosa is restricted to the xylem vessel environment, where mineral nutrients are transported through the plant host; therefore, changes in the concentrations of these elements likely impact the growth and virulence of this bacterium. Twitching motility, dependent on type IV pili (TFP), is required for movement against the transpiration stream that results in basipetal colonization. We previously demonstrated that calcium (Ca) increases the motility of X. fastidiosa, although the mechanism was unknown. PilY1 is a TFP structural protein recently shown to bind Ca and to regulate twitching and adhesion in bacterial pathogens of humans. Sequence analysis identified three pilY1 homologs in X. fastidiosa (PD0023, PD0502, and PD1611), one of which (PD1611) contains a Ca-binding motif. Separate deletions of PD0023 and PD1611 resulted in mutants that still showed twitching motility and were not impaired in attachment or biofilm formation. However, the response of increased twitching at higher Ca concentrations was lost in the pilY1-1611 mutant. Ca does not modulate the expression of any of the X. fastidiosa PilY1 homologs, although it increases the expression of the retraction ATPase pilT during active movement. The evidence presented here suggests functional differences between the PilY1 homologs, which may provide X. fastidiosa with an adaptive advantage in environments with high Ca concentrations, such as xylem sap. PMID:25217013

Calcium-Enhanced Twitching Motility in Xylella fastidiosa Is Linked to a Single PilY1 Homolog.

PubMed

Cruz, Luisa F; Parker, Jennifer K; Cobine, Paul A; De La Fuente, Leonardo

2014-12-01

The plant-pathogenic bacterium Xylella fastidiosa is restricted to the xylem vessel environment, where mineral nutrients are transported through the plant host; therefore, changes in the concentrations of these elements likely impact the growth and virulence of this bacterium. Twitching motility, dependent on type IV pili (TFP), is required for movement against the transpiration stream that results in basipetal colonization. We previously demonstrated that calcium (Ca) increases the motility of X. fastidiosa, although the mechanism was unknown. PilY1 is a TFP structural protein recently shown to bind Ca and to regulate twitching and adhesion in bacterial pathogens of humans. Sequence analysis identified three pilY1 homologs in X. fastidiosa (PD0023, PD0502, and PD1611), one of which (PD1611) contains a Ca-binding motif. Separate deletions of PD0023 and PD1611 resulted in mutants that still showed twitching motility and were not impaired in attachment or biofilm formation. However, the response of increased twitching at higher Ca concentrations was lost in the pilY1-1611 mutant. Ca does not modulate the expression of any of the X. fastidiosa PilY1 homologs, although it increases the expression of the retraction ATPase pilT during active movement. The evidence presented here suggests functional differences between the PilY1 homologs, which may provide X. fastidiosa with an adaptive advantage in environments with high Ca concentrations, such as xylem sap. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Characterization of Xylella fastidiosa lipopolysaccharide and its role in key steps of the disease cycle in grapevine

USDA-ARS?s Scientific Manuscript database

This project aims to elucidate molecular mechanisms of Xylella fastidiosa (Xf) pathogenicity. Work is focused on the lipopolysaccharide (LPS) component of the outer membrane, which consists of lipid A, core oligosaccharides, and a variable O-antigen moiety. Specifically, the O-antigen portion of LPS...

Plasmid transfer by conjugation as a possible route of horizontal gene transfer and recombination in Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Horizontal gene transfer is an important component of evolution and adaptation of bacterial species. Xylella fastidiosa has the ability to incorporate exogenous DNA into its genome by homologous recombination at relatively high rates. This genetic recombination is believed to play a role in adaptati...

Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1.

PubMed

Zhang, Shujian; Chakrabarty, Pranjib K; Fleites, Laura A; Rayside, Patricia A; Hopkins, Donald L; Gabriel, Dean W

2015-01-01

Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry.

Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1

PubMed Central

Zhang, Shujian; Chakrabarty, Pranjib K.; Fleites, Laura A.; Rayside, Patricia A.; Hopkins, Donald L.; Gabriel, Dean W.

2015-01-01

Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry. PMID:26218423

Association of Xylella fastidiosa with Yield Loss and Altered Fruit Quality in a Naturally Infected Rabbiteye Blueberry Orchard

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa causes disease in a number of plants in the southeastern United States, including southern highbush blueberry, but little was known concerning its potential impact in rabbiteye blueberry (Vaccinium virgatum). In a naturally infected orchard in Louisiana, mean yields of X. fastidi...

Seasonal fluctuations of sap-feeding insect species infected by Xylella fastidiosa in apulian olive groves of southern Italy

USDA-ARS?s Scientific Manuscript database

A study on seasonal abundance and infectivity by Xylella fastidiosa of Auchenorrhyncha species in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were mon...

The DinJ/RelE toxin-antitoxin system suppresses bacterial proliferation and virulence of Xylella fastidiosa in grapevine

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa, the causal agent of Pierce’s disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf scorching symptoms, senescence, and vine decline. It appears ...

Natural Competence and Recombination in the Plant Pathogen Xylella fastidiosa ▿

PubMed Central

Kung, Stephanie H.; Almeida, Rodrigo P. P.

2011-01-01

Homologous recombination is one of many forces contributing to the diversity, adaptation, and emergence of pathogens. For naturally competent bacteria, transformation is one possible route for the acquisition of novel genetic material. This study demonstrates that Xylella fastidiosa, a generalist bacterial plant pathogen responsible for many emerging plant diseases, is naturally competent and able to homologously recombine exogenous DNA into its genome. Several factors that affect transformation and recombination efficiencies, such as nutrient availability, growth stage, and methylation of transforming DNA, were identified. Recombination was observed in at least one out of every 106 cells when exogenous plasmid DNA was supplied and one out of every 107 cells when different strains were grown together in vitro. Based on previous genomic studies and experimental data presented here, there is mounting evidence that recombination can occur at relatively high rates and could play a large role in shaping the genetic diversity of X. fastidiosa. PMID:21666009

The distribution and biology of potential vectors of Xylella fastidiosa on coffee and citrus in Puerto Rico

USDA-ARS?s Scientific Manuscript database

Plant diseases caused by Xylella fastidiosa (Wells et al.) (Xf) surround the Caribbean Basin. Two major commodities of Puerto Rico, coffee and citrus, are highly susceptible to Xf. We surveyed potential vectors of Xf in coffee and citrus farms in western Puerto Rico over an 18 month period. Cicadel...

Global gene expression analysis of the heat shock response in the phytopathogen Xylella fastidiosa.

PubMed

Koide, Tie; Vêncio, Ricardo Z N; Gomes, Suely L

2006-08-01

Xylella fastidiosa is a phytopathogenic bacterium that is responsible for diseases in many economically important crops. Although different strains have been studied, little is known about X. fastidiosa stress responses. One of the better characterized stress responses in bacteria is the heat shock response, which induces the expression of specific genes to prevent protein misfolding and aggregation and to promote degradation of the irreversibly denatured polypeptides. To investigate X. fastidiosa genes involved in the heat shock response, we performed a whole-genome microarray analysis in a time course experiment. Globally, 261 genes were induced (9.7%) and 222 genes were repressed (8.3%). The expression profiles of the differentially expressed genes were grouped, and their expression patterns were validated by quantitative reverse transcription-PCR experiments. We determined the transcription start sites of six heat shock-inducible genes and analyzed their promoter regions, which allowed us to propose a putative consensus for sigma(32) promoters in Xylella and to suggest additional genes as putative members of this regulon. Besides the induction of classical heat shock protein genes, we observed the up-regulation of virulence-associated genes such as vapD and of genes for hemagglutinins, hemolysin, and xylan-degrading enzymes, which may indicate the importance of heat stress to bacterial pathogenesis. In addition, we observed the repression of genes related to fimbriae, aerobic respiration, and protein biosynthesis and the induction of genes related to the extracytoplasmic stress response and some phage-related genes, revealing the complex network of genes that work together in response to heat shock.

Detection and characterization of pXFSL21, a novel single-copy plasmid from Xylella fastidiosa Strain Stag’s Leap

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa Strain Stag’s Leap, originally isolated from Napa Valley, California, is highly virulent in causing Pierce’s Disease (PD) of grapevine. Plasmids are extrachromosomal genetic elements associated with bacterial environmental adaptation such as virulence development. In this study, t...

Draft genome sequence of Xylella fastidiosa supsp. multiplex strain Griffin-1 from Quercus rubra in Georgia

USDA-ARS?s Scientific Manuscript database

The draft genome sequence of Xylella fastidiosa subsp. multiplex Strain Griffin-1 isolated from a red oak tree (Quercus rubra) in Georgia, U.S.A. is reported. The bacterium has a genome size of 2,387,314 bp with 51.7% G+C content and comprises 2,903 predicted open reading frames (ORFs), and 50 RNA g...

Comparative genomic analysis of coffee-infecting Xylella fastidiosa strains isolated from Brazil.

PubMed

Barbosa, Deibs; Alencar, Valquíria Campos; Santos, Daiene Souza; de Freitas Oliveira, Ana Cláudia; de Souza, Alessandra A; Coletta-Filho, Helvecio D; de Oliveira, Regina Souza; Nunes, Luiz R

2015-05-01

Strains of Xylella fastidiosa constitute a complex group of bacteria that develop within the xylem of many plant hosts, causing diseases of significant economic importance, such as Pierce's disease in North American grapevines and citrus variegated chlorosis in Brazil. X. fastidiosa has also been obtained from other host plants, in direct correlation with the development of diseases, as in the case of coffee leaf scorch (CLS)--a disease with potential to cause severe economic losses to the Brazilian coffee industry. This paper describes a thorough genomic characterization of coffee-infecting X. fastidiosa strains, initially performed through a microarray-based approach, which demonstrated that CLS strains could be subdivided in two phylogenetically distinct subgroups. Whole-genomic sequencing of two of these bacteria (one from each subgroup) allowed identification of ORFs and horizontally transferred elements (HTEs) that were specific to CLS-related X. fastidiosa strains. Such analyses confirmed the size and importance of HTEs as major mediators of chromosomal evolution amongst these bacteria, and allowed identification of differences in gene content, after comparisons were made with previously sequenced X. fastidiosa strains, isolated from alternative hosts. Although direct experimentation still needs to be performed to elucidate the biological consequences associated with such differences, it was interesting to verify that CLS-related bacteria display variations in genes that produce toxins, as well as surface-related factors (such as fimbrial adhesins and LPS) that have been shown to be involved with recognition of specific host factors in different pathogenic bacteria. © 2015 The Authors.

Grapevine rootstock effects on scion sap phenolic levels, resistance to Xylella fastidiosa infection, and progression of Pierce’s disease

USDA-ARS?s Scientific Manuscript database

The xylem-limited bacterium Xylella fastidiosa (Xf) causes Pierce’s disease (PD), an important disease of grapevine, Vitis vinifera L. Grapevine rootstocks were developed to provide increased resistance to root disease, but rootstock effects on cane and vine diseases remain unclear. Grapevines that ...

Xylella fastidiosa esterase rather than hydroxynitrile lyase.

PubMed

Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

2015-03-02

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Radicinin from Cochliobolus sp. inhibits Xylella fastidiosa, the causal agent of Pierce's Disease of grapevine.

PubMed

Aldrich, Thomas J; Rolshausen, Philippe E; Roper, M Caroline; Reader, Jordan M; Steinhaus, Matthew J; Rapicavoli, Jeannette; Vosburg, David A; Maloney, Katherine N

2015-08-01

The fastidious phytopathogenic bacterium, Xylella fastidiosa, poses a substantial threat to many economically important crops, causing devastating diseases including Pierce's Disease of grapevine. Grapevines (Vitis vinifera L.) planted in an area under Pierce's Disease pressure often display differences in disease severity and symptom expression, with apparently healthy vines growing alongside the dying ones, despite the fact that all the vines are genetic clones of one another. Under the hypothesis that endophytic microbes might be responsible for this non-genetic resistance to X. fastidiosa, endophytic fungi were isolated from vineyard cvs. 'Chardonnay' and 'Cabernet Sauvignon' grown under high Pierce's Disease pressure. A Cochliobolus sp. isolated from a Cabernet Sauvignon grapevine inhibited the growth of X. fastidiosa in vitro. Bioassay-guided isolation of an organic extract of Cochliobolus sp. yielded the natural product radicinin as the major active compound. Radicinin also inhibited proteases isolated from the culture supernatant of X. fastidiosa. In order to assess structure-activity relationships, three semi-synthetic derivatives of radicinin were prepared and tested for activity against X. fastidiosa in vitro. Assay results of these derivatives are consistent with enzyme inactivation by conjugate addition to carbon-10 of radicinin, as proposed previously. Copyright © 2015 Elsevier Ltd. All rights reserved.

XatA, an AT-1 autotransporter important for the virulence of Xylella fastidiosa Temecula1

PubMed Central

Matsumoto, Ayumi; Huston, Sherry L; Killiny, Nabil; Igo, Michele M

2012-01-01

Xylella fastidiosa Temecula1 is the causative agent of Pierce's disease of grapevine, which is spread by xylem-feeding insects. An important feature of the infection cycle is the ability of X. fastidiosa to colonize and interact with two distinct environments, the xylem of susceptible plants and the insect foregut. Here, we describe our characterization of XatA, the X. fastidiosa autotransporter protein encoded by PD0528. XatA, which is classified as an AT-1 (classical) autotransporter, has a C-terminal β-barrel domain and a passenger domain composed of six tandem repeats of approximately 50 amino acids. Localization studies indicate that XatA is present in both the outer membrane and membrane vesicles and its passenger domain can be found in the supernatant. Moreover, XatA is important for X. fastidiosa autoaggregation and biofilm formation based on mutational analysis and the discovery that Escherichia coli expressing XatA acquire these traits. The xatA mutant also shows a significant decrease in Pierce's disease symptoms when inoculated into grapevines. Finally, X. fastidiosa homologs to XatA, which can be divided into three distinct groups based on synteny, form a single, well-supported clade, suggesting that they arose from a common ancestor. PMID:22950010

A Temperature-Independent Cold-Shock Protein Homolog Acts as a Virulence Factor in Xylella fastidiosa.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-05-01

Xylella fastidiosa, causal agent of Pierce's disease (PD) of grapevine, is a fastidious organism that requires very specific conditions for replication and plant colonization. Cold temperatures reduce growth and survival of X. fastidiosa both in vitro and in planta. However, little is known regarding physiological responses of X. fastidiosa to temperature changes. Cold-shock proteins (CSP), a family of nucleic acid-binding proteins, act as chaperones facilitating translation at low temperatures. Bacterial genomes often encode multiple CSP, some of which are strongly induced following exposure to cold. Additionally, CSP contribute to the general stress response through mRNA stabilization and posttranscriptional regulation. A putative CSP homolog (Csp1) with RNA-binding activity was identified in X. fastidiosa Stag's Leap. The csp1 gene lacked the long 5' untranslated region characteristic of cold-inducible genes and was expressed in a temperature-independent manner. As compared with the wild type, a deletion mutant of csp1 (∆csp1) had decreased survival rates following cold exposure and salt stress in vitro. The deletion mutant also was significantly less virulent in grapevine, as compared with the wild type, in the absence of cold stress. These results suggest an important function of X. fastidiosa Csp1 in response to cellular stress and during plant colonization.

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

PubMed Central

Baldi, Paolo; La Porta, Nicola

2017-01-01

In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific

Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

USDA-ARS?s Scientific Manuscript database

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

Diffusible signal factor (DSF) synthase RpfF of Xylella fastidiosa is a multifunction protein also required for response to DSF.

PubMed

Ionescu, Michael; Baccari, Clelia; Da Silva, Aline Maria; Garcia, Angelica; Yokota, Kenji; Lindow, Steven E

2013-12-01

Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes.

Diffusible Signal Factor (DSF) Synthase RpfF of Xylella fastidiosa Is a Multifunction Protein Also Required for Response to DSF

PubMed Central

Ionescu, Michael; Baccari, Clelia; Da Silva, Aline Maria; Garcia, Angelica; Yokota, Kenji

2013-01-01

Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes. PMID:24056101

RpfF-dependent regulon of Xylella fastidiosa.

PubMed

Wang, Nian; Li, Jian-Liang; Lindow, Steven E

2012-11-01

ABSTRACT Xylella fastidiosa regulates traits important to both virulence of grape as well as colonization of sharpshooter vectors via its production of a fatty acid signal molecule known as DSF whose production is dependent on rpfF. Although X. fastidiosa rpfF mutants exhibit increased virulence to plants, they are unable to be spread from plant to plant by insect vectors. To gain more insight into the traits that contribute to these processes, a whole-genome Agilent DNA microarray for this species was developed and used to determine the RpfF-dependent regulon by transcriptional profiling. In total, 446 protein coding genes whose expression was significantly different between the wild type and an rpfF mutant (false discovery rate < 0.05) were identified when cells were grown in PW liquid medium. Among them, 165 genes were downregulated in the rpfF mutant compared with the wild-type strain whereas 281 genes were over-expressed. RpfF function was required for regulation of 11 regulatory and σ factors, including rpfE, yybA, PD1177, glnB, rpfG, PD0954, PD0199, PD2050, colR, rpoH, and rpoD. In general, RpfF is required for regulation of genes involved in attachment and biofilm formation, enhancing expression of hemagglutinin genes hxfA and hxfB, and suppressing most type IV pili and gum genes. A large number of other RpfF-dependent genes that might contribute to virulence or insect colonization were also identified such as those encoding hemolysin and colicin V, as well as genes with unknown functions.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa.

PubMed

Fogaça, Andréa C; Zaini, Paulo A; Wulff, Nelson A; da Silva, Patrícia I P; Fázio, Marcos A; Miranda, Antônio; Daffre, Sirlei; da Silva, Aline M

2010-05-01

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

A chitinase is required for Xylella fastidiosa colonization of its insect and plant hosts.

PubMed

Labroussaa, Fabien; Ionescu, Michael; Zeilinger, Adam R; Lindow, Steven E; Almeida, Rodrigo P P

2017-04-01

Xylella fastidiosa colonizes the xylem network of host plant species as well as the foregut of its required insect vectors to ensure efficient propagation. Disease management strategies remain inefficient due to a limited comprehension of the mechanisms governing both insect and plant colonization. It was previously shown that X. fastidiosa has a functional chitinase (ChiA), and that chitin likely serves as a carbon source for this bacterium. We expand on that research, showing that a chiA mutant strain is unable to grow on chitin as the sole carbon source. Quantitative PCR assays allowed us to detect bacterial cells in the foregut of vectors after pathogen acquisition; populations of the wild-type and complemented mutant strain were both significantly larger than the chiA mutant strain 10 days, but not 3 days, post acquisition. These results indicate that adhesion of the chiA mutant strain to vectors may not be impaired, but that cell multiplication is limited. The mutant was also affected in its transmission by vectors to plants. In addition, the chiA mutant strain was unable to colonize host plants, suggesting that the enzyme has other substrates associated with plant colonization. Lastly, ChiA requires other X. fastidiosa protein(s) for its in vitro chitinolytic activity. The observation that the chiA mutant strain is not able to colonize plants warrants future attention to be paid to the substrates for this enzyme.

Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights.

PubMed

Bhattacharyya, Anamitra; Stilwagen, Stephanie; Reznik, Gary; Feil, Helene; Feil, William S; Anderson, Iain; Bernal, Axel; D'Souza, Mark; Ivanova, Natalia; Kapatral, Vinayak; Larsen, Niels; Los, Tamara; Lykidis, Athanasios; Selkov, Eugene; Walunas, Theresa L; Purcell, Alexander; Edwards, Rob A; Hawkins, Trevor; Haselkorn, Robert; Overbeek, Ross; Kyrpides, Nikos C; Predki, Paul F

2002-10-01

Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

Support for the salivation-egestion hypothesis for Xylella fastidiosa inoculation: salivary glucanase is injected into xylem during vector feeding.

USDA-ARS?s Scientific Manuscript database

The salivation-egestion hypothesis for the inoculation mechanism of Xylella fastidiosa (Xf) proposes that saliva secreted into plants is taken up into the vector’s precibarium. There, saliva loosens the Xf bacterial biofilm by enzymatically degrading ß-1, 4 glucans that form the chemical backbone o...

Analysis of the biofilm proteome of Xylella fastidiosa.

PubMed

Silva, Mariana S; De Souza, Alessandra A; Takita, Marco A; Labate, Carlos A; Machado, Marcos A

2011-09-22

Xylella fastidiosa is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of X. fastidiosa strain 9a5c, in comparison to planktonic growth condition. We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of X. fastidiosa. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments. We observed overexpression of proteins related to quorum sensing, proving the existence of

Analysis of the biofilm proteome of Xylella fastidiosa

PubMed Central

2011-01-01

Background Xylella fastidiosa is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of X. fastidiosa strain 9a5c, in comparison to planktonic growth condition. Results We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of X. fastidiosa. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments. Conclusions We observed overexpression of proteins related to quorum

The Major Outer Membrane Protein MopB Is Required for Twitching Movement and Affects Biofilm Formation and Virulence in Two Xylella fastidiosa strains.

PubMed

Chen, Hongyu; Kandel, Prem P; Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

2017-11-01

MopB is a major outer membrane protein (OMP) in Xylella fastidiosa, a bacterial plant pathogen that causes losses on many economically important crops. Based on in silico analysis, the uncharacterized MopB protein of X. fastidiosa contains a β-barrel structure with an OmpA-like domain and a predicted calcium-binding motif. Here, MopB function was studied by mutational analysis taking advantage of the natural competence of X. fastidiosa. Mutants of mopB were constructed in two different X. fastidiosa strains, the type strain Temecula and the more virulent WM1-1. Deletion of the mopB gene impaired cell-to-cell aggregation, surface attachment, and biofilm formation in both strains. Interestingly, mopB deletion completely abolished twitching motility. Electron microscopy of the bacterial cell surface revealed that mopB deletion eliminated type IV and type I pili formation, potentially caused by destabilization of the outer membrane. Both mopB mutants showed reduced virulence using tobacco (Nicotiana tabacum) as a host under greenhouse conditions. These results suggest that MopB has pleiotropic functions in biofilm formation and twitching motility and is important for virulence of X. fastidiosa.

Two whole genome sequences of Xylella fastidiosa (strains M12 and M23) causing almond leaf scorch disease in California

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a Gram negative, nutritionally fastidious plant pathogenic bacterium that causes many economically important diseases including almond leaf scorch disease (ALSD) and Pierce’s disease of grape in California, as well as citrus variegated chlorosis in South America. Genome inform...

Expression of pathogenicity-related genes of Xylella fastidiosa in vitro and in planta.

PubMed

de Souza, Alessandra A; Takita, Marco A; Pereira, Eridan O; Coletta-Filho, Helvécio D; Machado, Marcos A

2005-04-01

Xylella fastidiosa is responsible for several economically important plant diseases. It is currently assumed that the symptoms are caused by vascular occlusion due to biofilm formation. Microarray technology was previously used to examine the global gene expression profile of X. fastidiosa freshly isolated from symptomatic plants or after several passages by axenic culture medium, and different pathogenicity profiles have been obtained. In the present study the expression of some pathogenicity-related genes was evaluated in vitro and in planta by RT-PCR. The results suggest that adhesion is important at the beginning of biofilm formation, while the genes related to adaptation are essential for the organism's maintenance in planta. Similar results were observed in vitro mainly for the adhesion genes. The pattern of expression observed suggests that adhesion modulates biofilm formation whereas the expression of some adaptation genes may be related to the environment in which the organism is living.

Direct Evidence of Egestion and Salivation of Xylella fastidiosa Suggests Sharpshooters Can Be "Flying Syringes".

PubMed

Backus, Elaine A; Shugart, Holly J; Rogers, Elizabeth E; Morgan, J Kent; Shatters, Robert

2015-05-01

Xylella fastidiosa is unique among insect-transmitted plant pathogens because it is propagative but noncirculative, adhering to and multiplying on the cuticular lining of the anterior foregut. Any inoculation mechanism for X. fastidiosa must explain how bacterial cells exit the vector's stylets via the food canal and directly enter the plant. A combined egestion-salivation mechanism has been proposed to explain these unique features. Egestion is the putative outward flow of fluid from the foregut via hypothesized bidirectional pumping of the cibarium. The present study traced green fluorescent protein-expressing X. fastidiosa or fluorescent nanoparticles acquired from artificial diets by glassy-winged sharpshooters, Homalodisca vitripennis, as they were egested into simultaneously secreted saliva. X. fastidiosa or nanoparticles were shown to mix with gelling saliva to form fluorescent deposits and salivary sheaths on artificial diets, providing the first direct, conclusive evidence of egestion by any hemipteran insect. Therefore, the present results strongly support an egestion-salivation mechanism of X. fastidiosa inoculation. Results also support that a column of fluid is transiently held in the foregut without being swallowed. Evidence also supports (but does not definitively prove) that bacteria were suspended in the column of fluid during the vector's transit from diet to diet, and were egested with the held fluid. Thus, we hypothesize that sharpshooters could be true "flying syringes," especially when inoculation occurs very soon after uptake of bacteria, suggesting the new paradigm of a nonpersistent X. fastidiosa transmission mechanism.

Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa.

PubMed

Marques, M V; da Silva, A M; Gomes, S L

2001-05-01

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.

Expression of putative pathogenicity-related genes in Xylella fastidiosa grown at low and high cell density conditions in vitro.

PubMed

Scarpari, Leandra M; Lambais, Marcio R; Silva, Denise S; Carraro, Dirce M; Carrer, Helaine

2003-05-16

Xylella fastidiosa is the causal agent of economically important plant diseases, including citrus variegated chlorosis and Pierce's disease. Hitherto, there has been no information on the molecular mechanisms controlling X. fastidiosa-plant interactions. To determine whether predicted open reading frames (ORFs) encoding putative pathogenicity-related factors were expressed by X. fastidiosa 9a5c cells grown at low (LCD) and high cell density (HCD) conditions in liquid modified PW medium, reverse Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed. Our results indicated that ORFs XF2344, XF2369, XF1851 and XF0125, encoding putative Fur, GumC, a serine-protease and RsmA, respectively, were significantly suppressed at HCD conditions. In contrast, ORF XF1115, encoding putative RpfF, was significantly induced at HCD conditions. Expressions of ORFs XF2367, XF2362 and XF0290, encoding putative GumD, GumJ and RpfA, respectively, were detected only at HCD conditions, whereas expression of ORF XF0287, encoding putative RpfB was detected only at LCD conditions. Bioassays with an Agrobacterium traG::lacZ reporter system indicated that X. fastidiosa does not synthesize N-acyl-homoserine lactones, whereas bioassays with a diffusible signal factor (DSF)-responsive Xanthomonas campestris pv. campestris mutant indicate that X. fastidiosa synthesizes a molecule similar to DSF in modified PW medium. Our data also suggest that the synthesis of the DSF-like molecule and fastidian gum by X. fastidiosa is affected by cell density in vitro.

De Novo whole genome sequence of Xylella fastidiosa subsp. multiplex strain BB01 from blueberry in Georgia, USA

USDA-ARS?s Scientific Manuscript database

This study reports a de novo assembled draft genome sequence of Xylella fastidiosa subsp. multiplex strain BB01 causing blueberry bacterial leaf scorch in Georgia, USA. The BB01 genome is 2,517,579 bp with a G+C content of 51.8% and 2,943 open reading frames (ORFs) and 48 RNA genes....

The diversity of citrus endophytic bacteria and their interactions with Xylella fastidiosa and host plants.

PubMed

Azevedo, João Lúcio; Araújo, Welington Luiz; Lacava, Paulo Teixeira

2016-01-01

The bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC) and has been associated with important losses in commercial orchards of all sweet orange [Citrus sinensis (L.)] cultivars. The development of this disease depends on the environmental conditions, including the endophytic microbial community associated with the host plant. Previous studies have shown that X. fastidiosa interacts with the endophytic community in xylem vessels as well as in the insect vector, resulting in a lower bacterial population and reduced CVC symptoms. The citrus endophytic bacterium Methylobacterium mesophilicum can trigger X. fastidiosa response in vitro, which results in reduced growth and induction of genes associated with energy production, stress, transport, and motility, indicating that X. fastidiosa has an adaptive response to M. mesophilicum. Although this response may result in reduced CVC symptoms, the colonization rate of the endophytic bacteria should be considered in studies that intend to use this endophyte to suppress CVC disease. Symbiotic control is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace pathogens. Candidate endophytes for symbiotic control of CVC must occupy the xylem of host plants and attach to the precibarium of sharpshooter insects to access the pathogen. In the present review, we focus on interactions between endophytic bacteria from sweet orange plants and X. fastidiosa, especially those that may be candidates for control of CVC.

The diversity of citrus endophytic bacteria and their interactions with Xylella fastidiosa and host plants

PubMed Central

Azevedo, João Lúcio; Araújo, Welington Luiz; Lacava, Paulo Teixeira

2016-01-01

Abstract The bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC) and has been associated with important losses in commercial orchards of all sweet orange [Citrus sinensis (L.)] cultivars. The development of this disease depends on the environmental conditions, including the endophytic microbial community associated with the host plant. Previous studies have shown that X. fastidiosa interacts with the endophytic community in xylem vessels as well as in the insect vector, resulting in a lower bacterial population and reduced CVC symptoms. The citrus endophytic bacterium Methylobacterium mesophilicum can trigger X. fastidiosa response in vitro, which results in reduced growth and induction of genes associated with energy production, stress, transport, and motility, indicating that X. fastidiosa has an adaptive response to M. mesophilicum. Although this response may result in reduced CVC symptoms, the colonization rate of the endophytic bacteria should be considered in studies that intend to use this endophyte to suppress CVC disease. Symbiotic control is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace pathogens. Candidate endophytes for symbiotic control of CVC must occupy the xylem of host plants and attach to the precibarium of sharpshooter insects to access the pathogen. In the present review, we focus on interactions between endophytic bacteria from sweet orange plants and X. fastidiosa, especially those that may be candidates for control of CVC. PMID:27727362

Fractal analysis of Xylella fastidiosa biofilm formation

NASA Astrophysics Data System (ADS)

Moreau, A. L. D.; Lorite, G. S.; Rodrigues, C. M.; Souza, A. A.; Cotta, M. A.

2009-07-01

We have investigated the growth process of Xylella fastidiosa biofilms inoculated on a glass. The size and the distance between biofilms were analyzed by optical images; a fractal analysis was carried out using scaling concepts and atomic force microscopy images. We observed that different biofilms show similar fractal characteristics, although morphological variations can be identified for different biofilm stages. Two types of structural patterns are suggested from the observed fractal dimensions Df. In the initial and final stages of biofilm formation, Df is 2.73±0.06 and 2.68±0.06, respectively, while in the maturation stage, Df=2.57±0.08. These values suggest that the biofilm growth can be understood as an Eden model in the former case, while diffusion-limited aggregation (DLA) seems to dominate the maturation stage. Changes in the correlation length parallel to the surface were also observed; these results were correlated with the biofilm matrix formation, which can hinder nutrient diffusion and thus create conditions to drive DLA growth.

Xylella fastidiosa in rabbiteye blueberry in Louisiana is genetically similar to a strain found in Southern highbush blueberry in Georgia

USDA-ARS?s Scientific Manuscript database

During the past ten years, Xylella fastidiosa has been confirmed as a pathogen of Southern highbush blueberry (Vaccinium corymbosum interspecific hybrids) in Georgia and Florida. Recent work in Louisiana has shown that it is also associated with reduced yield and altered fruit quality in ‘Tifblue’ ...

Differentiation of Xylella fastidiosa Strains via Multilocus Sequence Analysis of Environmentally Mediated Genes (MLSA-E)

PubMed Central

Parker, Jennifer K.; Havird, Justin C.

2012-01-01

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing

Differentiation of Xylella fastidiosa strains via multilocus sequence analysis of environmentally mediated genes (MLSA-E).

PubMed

Parker, Jennifer K; Havird, Justin C; De La Fuente, Leonardo

2012-03-01

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing

TolC is required for pathogenicity of Xylella fastidiosa in Vitis vinifera grapevines.

PubMed

Reddy, Joseph D; Reddy, Stephanie L; Hopkins, Don L; Gabriel, Dean W

2007-04-01

Xylella fastidiosa infects a wide range of hosts and causes serious diseases on some of them. The complete genomic sequences of both a citrus variegated chlorosis (CVC) and a Pierce's disease (PD) strain revealed two type I protein secretion plus two multidrug resistance efflux systems, and all evidently were dependent on a single tolC homolog. Marker exchange mutagenesis of the single tolC gene in PD strain Temecula resulted in a total loss of pathogenicity on grape. Importantly, the tolC- mutant strains were not recovered after inoculation into grape xylem, strongly indicating that multidrug efflux is critical to survival of this fastidious pathogen. Both survival and pathogenicity were restored by complementation using tolC cloned in shuttle vector pBBR1MCS-5, which was shown to replicate autonomously, without selection, for 60 days in Temecula growing in planta. These results also demonstrate the ability to complement mutations in X. fastidiosa.

Detection of the Bacterium, Xylella fastidiosa, in Saliva of Glassy-Winged Sharpshooter, Homalodisca vitripennis

PubMed Central

Ramirez, Jose L.; Lacava, Paulo T.; Miller, Thomas A.

2008-01-01

Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), the glassy-winged sharpshooter, is one of the most important vectors of the bacterium, Xylella fastidiosa subsp. piercei (Xanthomonadales: Xanthomonadaceae) that causes Pierce's Disease in grapevines in California. In the present study we report a new method for studying pathogen transmission or probing behavior of H. vitripennis. When confined, H. vitripennis attempt to probe the surface of sterile containers 48 hours post-acquisition of X. f. piercei. The saliva deposited during attempted feeding probes was found to contain X. f. piercei. We observed no correlation between X. f. piercei titers in the foregut of H. vitripennis that fed on Xylella-infected grapevines and the presence of this bacterium in the deposited saliva. The infection rate after a 48 h post-acquisition feeding on healthy citrus and grapevines was observed to be 77% for H. vitripennis that fed on grapevines and 81% for H. vitripennis that fed on citrus, with no difference in the number of positive probing sites from H. vitripennis that fed on either grapevine or citrus. This method is amenable for individual assessment of X. f. piercei-infecuvity, with samples less likely to be affected by tissue contamination that is usually present in whole body extracts. PMID:20233080

Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

PubMed

Oliver, J E; Sefick, S A; Parker, J K; Arnold, T; Cobine, P A; De La Fuente, L

2014-10-01

Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.

Population Structure of Xylella fastidiosa Associated with Almond Leaf Scorch Disease in the San Joaquin Valley of California.

PubMed

Lin, Hong; Islam, Md Sajedul; Cabrera-La Rosa, Juan C; Civerolo, Edwin L; Groves, Russell L

2015-06-01

Xylella fastidiosa causes disease in many commercial crops, including almond leaf scorch (ALS) disease in susceptible almond (Prunus dulcis). In this study, genetic diversity and population structure of X. fastidiosa associated with ALS disease were evaluated. Isolates obtained from two almond orchards in Fresno and Kern County in the San Joaquin Valley of California were analyzed for two successive years. Multilocus simple-sequence repeat (SSR) analysis revealed two major genetic clusters that were associated with two host cultivars, 'Sonora' and 'Nonpareil', respectively, regardless of the year of study or location of the orchard. These relationships suggest that host cultivar selection and adaptation are major driving forces shaping ALS X. fastidiosa population structure in the San Joaquin Valley. This finding will provide insight into understanding pathogen adaptation and host selection in the context of ALS disease dynamics.

Transcription analysis of pilS and xpsEL genes from Xylella fastidiosa.

PubMed

Coltri, Patricia P; Rosato, Yoko B

2005-04-01

Xylella fastidiosa is a xylem-limited phytopathogen responsible for diseases in several plants such as citrus and coffee. Analysis of the bacterial genome revealed some putative pathogenicity-related genes that could help to elucidate the molecular mechanisms of plant-pathogen interactions. In the present work, the transcription of three genes of the bacterium, grown in defined and rich media and also in media containing host plant extracts (sweet orange, 'ponkan' and coffee) was analyzed by RT-PCR. The pilS gene, which encodes a sensor histidine kinase responsible for the biosynthesis of fimbriae, was transcribed when the bacterium was grown in more complex media such as PW and in medium containing plant extracts. The xps genes (xpsL and xpsE) which are related to the type II secretion system were also detected when the bacterium was grown in rich media and media with 'ponkan' and coffee extracts. It was thus observed that pilS and xpsEL genes of X. fastidiosa can be modulated by environmental factors and their expression is dependent on the nutritional status of the growth medium.

Effects of Grape Xylem Sap and Cell-Wall Constituents on In Vitro Growth, Biofilm Formation and Cellular Aggregation of Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Purified cell-wall constituents or grape xylem sap added to media affected in vitro growth, biofilm formation, cell aggregation and gene expression of Xylella fastidiosa. Media containing xylem sap from Pierce’s disease (PD)-susceptible plants provided better support for bacterial growth and biofil...

Homologous Recombination and Xylella fastidiosa Host-Pathogen Associations in South America.

PubMed

Coletta-Filho, Helvécio D; Francisco, Carolina S; Lopes, João R S; Muller, Christiane; Almeida, Rodrigo P P

2017-03-01

Homologous recombination affects the evolution of bacteria such as Xylella fastidiosa, a naturally competent plant pathogen that requires insect vectors for dispersal. This bacterial species is taxonomically divided into subspecies, with phylogenetic clusters within subspecies that are host specific. One subspecies, pauca, is primarily limited to South America, with the exception of recently reported strains in Europe and Costa Rica. Despite the economic importance of X. fastidiosa subsp. pauca in South America, little is known about its genetic diversity. Multilocus sequence typing (MLST) has previously identified six sequence types (ST) among plant samples collected in Brazil (both subsp. pauca and multiplex). Here, we report on a survey of X. fastidiosa genetic diversity (MLST based) performed in six regions in Brazil and two in Argentina, by sampling five different plant species. In addition to the six previously reported ST, seven new subsp. pauca and two new subsp. multiplex ST were identified. The presence of subsp. multiplex in South America is considered to be the consequence of a single introduction from its native range in North America more than 80 years ago. Different phylogenetic approaches clustered the South American ST into four groups, with strains infecting citrus (subsp. pauca); coffee and olive (subsp. pauca); coffee, hibiscus, and plum (subsp. pauca); and plum (subsp. multiplex). In areas where these different genetic clusters occurred sympatrically, we found evidence of homologous recombination in the form of bidirectional allelic exchange between subspp. pauca and multiplex. In fact, the only strain of subsp. pauca isolated from a plum host had an allele that originated from subsp. multiplex. These signatures of bidirectional homologous recombination between endemic and introduced ST indicate that gene flow occurs in short evolutionary time frames in X. fastidiosa, despite the ecological isolation (i.e., host plant species) of genotypes.

Phenology of Xylella fastidiosa and Draeculocephala minerva in California almond nurseries: an assessment of plant vulnerability to almond leaf scorch disease

USDA-ARS?s Scientific Manuscript database

Management of a plant disease requires knowledge of all possible infection pathways. Almond leaf scorch disease (ALSD) is caused by the xylem-limited bacterium Xylella fastidiosa, which is transmitted by several species of leafhoppers. The objectives of this research were to elucidate the fate of b...

Characterization of the Xylella fastidiosa PD1311 gene mutant and its suppression of Pierce's disease on grapevines.

PubMed

Hao, Lingyun; Johnson, Kameka; Cursino, Luciana; Mowery, Patricia; Burr, Thomas J

2017-06-01

Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl-coenzyme A (acyl-CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H 2 O 2 and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild-type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1-only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease. © 2016 BSPP AND JOHN WILEY & SONS LTD.

PemK toxin encoded by the Xylella fastidiosa IncP-1 plasmid pXF-RIV11 is a ribonuclease

USDA-ARS?s Scientific Manuscript database

Stable inheritance of the IncP-1 plasmid pXF-RIV11 in Xylella fastidiosa is conferred by the pemI/pemK plasmid addiction system. PemK serves as a toxin inhibiting bacterial growth; PemI is the corresponding antitoxin that blocks activity of PemK toxin by direct binding. Here, PemK toxin and PemI ant...

Site-Directed Disruption of the fimA and fimF Fimbrial Genes of Xylella fastidiosa.

PubMed

Feil, Helene; Feil, William S; Detter, John C; Purcel, Alexander H; Lindow, Steven E

2003-06-01

ABSTRACT Xylella fastidiosa causes Pierce's disease, a serious disease of grape, citrus variegated chlorosis, almond and oleander leaf scorches, and many other similar diseases. Although the complete genome sequences of several strains of this organism are now available, the function of most genes in this organism, especially those conferring virulence, is lacking. Attachment of X. fastidiosa to xylem vessels and insect vectors may be required for virulence and transmission; therefore, we disrupted fimA and fimF, genes encoding the major fimbrial protein FimA and a homolog of the fimbrial adhesin MrkD, to determine their role in the attachment process. Disruption of the fimA and fimF genes in Temecula1 and STL grape strains of X. fastidiosa was obtained by homologous recombination using plasmids pFAK and pFFK, respectively. These vectors contained a kanamycin resistance gene cloned into either the fimA or fimF genes of X. fastidiosa grape strains Temecula1 or STL. Efficiency of transformation was sufficiently high ( approximately 600 transformants per mug of pFFK DNA) to enable selection of rare recombination events. Polymerase chain reaction and Southern blot analyses of the mutants indicated that a double crossover event had occurred exclusively within the fimA and fimF genes, replacing the chromosomal gene with the disrupted gene and abolishing production of the corresponding proteins, FimA or FimF. Scanning electron microscopy revealed that fimbriae size and number, cell aggregation, and cell size were reduced for the FimA or FimF mutants of X. fastidiosa when compared with the parental strain. FimA or FimF mutants of X. fastidiosa remained pathogenic to grapevines, with bacterial populations slightly reduced compared with those of the wild-type X. fastidiosa cells. These mutants maintained their resistance to kanamycin in planta for at least 6 months in the greenhouse.

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces.

PubMed

Ionescu, Michael; Zaini, Paulo A; Baccari, Clelia; Tran, Sophia; da Silva, Aline M; Lindow, Steven E

2014-09-16

Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an "exploratory" lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents.

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces

PubMed Central

Ionescu, Michael; Zaini, Paulo A.; Baccari, Clelia; Tran, Sophia; da Silva, Aline M.; Lindow, Steven E.

2014-01-01

Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an “exploratory” lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents. PMID:25197068

Transmission efficiency of Xylella fastidiosa by sharpshooters (Hemiptera: Cicadellidae) in coffee and citrus.

PubMed

Marucci, Rosangela C; Lopes, João R S; Cavichioli, Rodney R

2008-08-01

Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) is a bacterial pathogen transmitted by several sharpshooters in two tribes of Cicadellinae (Proconiini and Cicadellini). Here, we compared the transmission efficiency of X. fastidiosa in coffee (Coffea arabica L.) and citrus [Citrus sinensis (L.) Osbeck] by Cicadellini [Bucephalogonia xanthophis (Berg) and Dilobopterus costalimai Young] and Proconiini [Homalodisca ignorata Melichar and Oncometopia facialis (Signoret)] sharpshooters that occur in both crops. At different seasons, healthy adults of each species were submitted to a 48-h acquisition access period on citrus or coffee source plants infected with X. fastidiosa isolates that cause Citrus variegated chlorosis (CVC) and Coffee leaf scorch (CLS), respectively, and then confined on healthy seedlings of the corresponding host plant for a 48-h inoculation access period. No significant effect of inoculation season was observed when comparing infection rates of citrus or coffee plants inoculated by vectors at different times of the year. In citrus, the transmission rate by single insects was significantly higher for H. ignorata (30%) in relation to B. xanthophis (5%) and O. facialis (1.1%), but there was no difference among vector species in coffee, whose transmission rates ranged from 1.2 to 7.2%. Comparing host plants, H. ignorata was more effective in transmitting X. fastidiosa to citrus (30%) in relation to coffee (2.2%), whereas the other vectors transmitted the bacterium to both hosts with similar efficiencies. Despite these variations, vector efficiency in coffee and citrus is lower than that reported in other hosts.

Xylella fastidiosa differentially accumulates mineral elements in biofilm and planktonic cells.

PubMed

Cobine, Paul A; Cruz, Luisa F; Navarrete, Fernando; Duncan, Daniel; Tygart, Melissa; De La Fuente, Leonardo

2013-01-01

Xylella fastidiosa is a bacterial plant pathogen that infects numerous plant hosts. Disease develops when the bacterium colonizes the xylem vessels and forms a biofilm. Inductively coupled plasma optical emission spectroscopy was used to examine the mineral element content of this pathogen in biofilm and planktonic states. Significant accumulations of copper (30-fold), manganese (6-fold), zinc (5-fold), calcium (2-fold) and potassium (2-fold) in the biofilm compared to planktonic cells were observed. Other mineral elements such as sodium, magnesium and iron did not significantly differ between biofilm and planktonic cells. The distribution of mineral elements in the planktonic cells loosely mirrors the media composition; however the unique mineral element distribution in biofilm suggests specific mechanisms of accumulation from the media. A cell-to-surface attachment assay shows that addition of 50 to 100 µM Cu to standard X. fastidiosa media increases biofilm, while higher concentrations (>200 µM) slow cell growth and prevent biofilm formation. Moreover cell-to-surface attachment was blocked by specific chelation of copper. Growth of X. fastidiosa in microfluidic chambers under flow conditions showed that addition of 50 µM Cu to the media accelerated attachment and aggregation, while 400 µM prevented this process. Supplementation of standard media with Mn showed increased biofilm formation and cell-to-cell attachment. In contrast, while the biofilm accumulated Zn, supplementation to the media with this element caused inhibited growth of planktonic cells and impaired biofilm formation. Collectively these data suggest roles for these minerals in attachment and biofilm formation and therefore the virulence of this pathogen.

Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity.

PubMed

Scally, Mark; Schuenzel, Erin L; Stouthamer, Richard; Nunney, Leonard

2005-12-01

Multilocus sequence typing (MLST) identifies and groups bacterial strains based on DNA sequence data from (typically) seven housekeeping genes. MLST has also been employed to estimate the relative contributions of recombination and point mutation to clonal divergence. We applied MLST to the plant pathogen Xylella fastidiosa using an initial set of sequences for 10 loci (9.3 kb) of 25 strains from five different host plants, grapevine (PD strains), oleander (OLS strains), oak (OAK strains), almond (ALS strains), and peach (PP strains). An eBURST analysis identified six clonal complexes using the grouping criterion that each member must be identical to at least one other member at 7 or more of the 10 loci. These clonal complexes corresponded to previously identified phylogenetic clades; clonal complex 1 (CC1) (all PD strains plus two ALS strains) and CC2 (OLS strains) defined the X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi clades, while CC3 (ALS strains), CC4 (OAK strains), and CC5 (PP strains) were subclades of X. fastidiosa subsp. multiplex. CC6 (ALS strains) identified an X. fastidiosa subsp. multiplex-like group characterized by a high frequency of intersubspecific recombination. Compared to the recombination rate in other bacterial species, the recombination rate in X. fastidiosa is relatively low. Recombination between different alleles was estimated to give rise to 76% of the nucleotide changes and 31% of the allelic changes observed. The housekeeping loci holC, nuoL, leuA, gltT, cysG, petC, and lacF were chosen to form the basis of a public database for typing X. fastidiosa (www.mlst.net). These loci identified the same six clonal complexes using the strain grouping criterion of identity at five or more loci with at least one other member.

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

PubMed

Rogers, Elizabeth E; Stenger, Drake C

2012-01-01

A ≈ 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.

A Conjugative 38 kB Plasmid Is Present in Multiple Subspecies of Xylella fastidiosa

PubMed Central

Rogers, Elizabeth E.; Stenger, Drake C.

2012-01-01

A ∼38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19th century. PMID:23251694

Detection and analysis of the bacterium, Xylella fastidiosa, in glassy-winged sharpshooter, Homalodisca vitripennis, populations in Texas.

PubMed

Hail, Daymon; Mitchell, Forrest; Lauzière, Isabelle; Marshall, Patrick; Brady, Jeff; Bextine, Blake

2010-01-01

The glassy-winged sharpshooter, Homalodisca vitripeninis Germar (Hemiptera: Cicadellidae), is a xylophagous insect that is an endemic pest of several economically important plants in Texas. H. vitripennis is the main vector of Xylella fastidiosa Wells (Xanthomonadales: Xanthomonadaceae), the bacterium that causes Pierce's disease of grapevine and can travel long distances putting much of Texas grape production at risk. Understanding the movement of H. vitripennis populations capable of transmitting X. fastidiosa into Pierce's-disease-free areas is critical for developing a management program for Pierce's disease. To that end, the USDA-APHIS has developed a program to sample vineyards across Texas to monitor populations of H. vitripennis. From this sampling, H vitripennis collected during 2005 and 2006 over the months of May, June, and July from eight vineyards in different regions of Texas were recovered from yellow sticky traps and tested for the presence of X. fastidiosa. The foregut contents were vacuum extracted and analyzed using RT-PCR to determine the percentage of H. vitripennis within each population that harbor X. fastidiosa and have the potential to transmit this pathogen. H. vitripennis from vineyards known to have Pierce's disease routinely tested positive for the presence of X. fastidiosa. While almost all H. vitripennis collected from vineyards with no history of Pierce's disease tested negative for the presence of the pathogen, three individual insects tested positive. Furthermore, all three insects were determined, by DNA sequencing, to be carrying a strain of X. fastidiosa homologous to known Pierce's disease strains, signifying them as a risk factor for new X. fastidiosa infections.

Detection and Analysis of the Bacterium, Xylella fastidiosa, in Glassy-Winged Sharpshooter, Homalodisca vitripennis, Populations in Texas

PubMed Central

Hail, Daymon; Mitchell, Forrest; Lauzière, Isabelle; Marshall, Patrick; Brady, Jeff; Bextine, Blake

2010-01-01

The glassy-winged sharpshooter, Homalodisca vitripeninis Germar (Hemiptera: Cicadellidae), is a xylophagous insect that is an endemic pest of several economically important plants in Texas. H. vitripennis is the main vector of Xylella fastidiosa Wells (Xanthomonadales: Xanthomonadaceae), the bacterium that causes Pierce's disease of grapevine and can travel long distances putting much of Texas grape production at risk. Understanding the movement of H. vitripennis populations capable of transmitting X. fastidiosa into Pierce's-disease-free areas is critical for developing a management program for Pierce's disease. To that end, the USDA-APHIS has developed a program to sample vineyards across Texas to monitor populations of H. vitripennis. From this sampling, H vitripennis collected during 2005 and 2006 over the months of May, June, and July from eight vineyards in different regions of Texas were recovered from yellow sticky traps and tested for the presence of X. fastidiosa. The foregut contents were vacuum extracted and analyzed using RT-PCR to determine the percentage of H. vitripennis within each population that harbor X. fastidiosa and have the potential to transmit this pathogen. H. vitripennis from vineyards known to have Pierce's disease routinely tested positive for the presence of X. fastidiosa. While almost all H. vitripennis collected from vineyards with no history of Pierce's disease tested negative for the presence of the pathogen, three individual insects tested positive. Furthermore, all three insects were determined, by DNA sequencing, to be carrying a strain of X. fastidiosa homologous to known Pierce's disease strains, signifying them as a risk factor for new X. fastidiosa infections. PMID:21062210

Promiscuous Diffusible Signal Factor Production and Responsiveness of the Xylella fastidiosa Rpf System.

PubMed

Ionescu, Michael; Yokota, Kenji; Antonova, Elena; Garcia, Angelica; Beaulieu, Ellen; Hayes, Terry; Iavarone, Anthony T; Lindow, Steven E

2016-07-19

Cell density-dependent regulation of gene expression in Xylella fastidiosa that is crucial to its switching between plant hosts and insect vectors is dependent on RpfF and its production of 2-enoic acids known as diffusible signal factor (DSF). We show that X. fastidiosa produces a particularly large variety of similar, relatively long-chain-length 2-enoic acids that are active in modulating gene expression. Both X. fastidiosa itself and a Pantoea agglomerans surrogate host harboring X. fastidiosa RpfF (XfRpfF) is capable of producing a variety of both saturated and unsaturated free fatty acids. However, only 2-cis unsaturated acids were found to be biologically active in X. fastidiosa X. fastidiosa produces, and is particularly responsive to, a novel DSF species, 2-cis-hexadecanoic acid that we term XfDSF2. It is also responsive to other, even longer 2-enoic acids to which other taxa such as Xanthomonas campestris are unresponsive. The 2-enoic acids that are produced by X. fastidiosa are strongly affected by the cellular growth environment, with XfDSF2 not detected in culture media in which 2-tetradecenoic acid (XfDSF1) had previously been found. X. fastidiosa is responsive to much lower concentrations of XfDSF2 than XfDSF1. Apparently competitive interactions can occur between various saturated and unsaturated fatty acids that block the function of those agonistic 2-enoic fatty acids. By altering the particular 2-enoic acids produced and the relative balance of free enoic and saturated fatty acids, X. fastidiosa might modulate the extent of DSF-mediated quorum sensing. X. fastidiosa, having a complicated lifestyle in which it moves and multiplies within plants but also must be vectored by insects, utilizes DSF-based quorum sensing to partition the expression of traits needed for these two processes within different cells in this population based on local cellular density. The finding that it can produce a variety of DSF species in a strongly

Natural Competence of Xylella fastidiosa Occurs at a High Frequency Inside Microfluidic Chambers Mimicking the Bacterium's Natural Habitats.

PubMed

Kandel, Prem P; Lopez, Samantha M; Almeida, Rodrigo P P; De La Fuente, Leonardo

2016-09-01

Xylella fastidiosa is a xylem-limited bacterium that is the causal agent of emerging diseases in a number of economically important crops. Genetic diversity studies have demonstrated homologous recombination occurring among X. fastidiosa strains, which has been proposed to contribute to host plant shifts. Moreover, experimental evidence confirmed that X. fastidiosa is naturally competent for recombination in vitro Here, as an approximation of natural habitats (plant xylem vessels and insect mouthparts), recombination was studied in microfluidic chambers (MCs) filled with media amended with grapevine xylem sap. First, different media were screened for recombination in solid agar plates using a pair of X. fastidiosa strains that were previously reported to recombine in coculture. The highest frequency of recombination was obtained with PD3 medium, compared to those with the other two media (X. fastidiosa medium [XFM] and periwinkle wilt [PW] medium) used in previous studies. Dissection of the media components led to the identification of bovine serum albumin as an inhibitor of recombination that was correlated to its previously known effect on inhibition of twitching motility. When recombination was performed in liquid culture, the frequencies were significantly higher under flow conditions (MCs) than under batch conditions (test tubes). The recombination frequencies in MCs and agar plates were not significantly different from each other. Grapevine xylem sap from both susceptible and tolerant varieties allowed high recombination frequency in MCs when mixed with PD3. These results suggest that X. fastidiosa has the ability to be naturally competent in the natural growth environment of liquid flow, and this phenomenon could have implications in X. fastidiosa environmental adaptation. Xylella fastidiosa is a plant pathogen that lives inside xylem vessels (where water and nutrients are transported inside the plant) and the mouthparts of insect vectors. This bacterium

Natural Competence of Xylella fastidiosa Occurs at a High Frequency Inside Microfluidic Chambers Mimicking the Bacterium's Natural Habitats

PubMed Central

Kandel, Prem P.; Lopez, Samantha M.; Almeida, Rodrigo P. P.

2016-01-01

ABSTRACT Xylella fastidiosa is a xylem-limited bacterium that is the causal agent of emerging diseases in a number of economically important crops. Genetic diversity studies have demonstrated homologous recombination occurring among X. fastidiosa strains, which has been proposed to contribute to host plant shifts. Moreover, experimental evidence confirmed that X. fastidiosa is naturally competent for recombination in vitro. Here, as an approximation of natural habitats (plant xylem vessels and insect mouthparts), recombination was studied in microfluidic chambers (MCs) filled with media amended with grapevine xylem sap. First, different media were screened for recombination in solid agar plates using a pair of X. fastidiosa strains that were previously reported to recombine in coculture. The highest frequency of recombination was obtained with PD3 medium, compared to those with the other two media (X. fastidiosa medium [XFM] and periwinkle wilt [PW] medium) used in previous studies. Dissection of the media components led to the identification of bovine serum albumin as an inhibitor of recombination that was correlated to its previously known effect on inhibition of twitching motility. When recombination was performed in liquid culture, the frequencies were significantly higher under flow conditions (MCs) than under batch conditions (test tubes). The recombination frequencies in MCs and agar plates were not significantly different from each other. Grapevine xylem sap from both susceptible and tolerant varieties allowed high recombination frequency in MCs when mixed with PD3. These results suggest that X. fastidiosa has the ability to be naturally competent in the natural growth environment of liquid flow, and this phenomenon could have implications in X. fastidiosa environmental adaptation. IMPORTANCE Xylella fastidiosa is a plant pathogen that lives inside xylem vessels (where water and nutrients are transported inside the plant) and the mouthparts of insect

Detecting genetic introgression: high levels of intersubspecific recombination found in Xylella fastidiosa in Brazil.

PubMed

Nunney, Leonard; Yuan, Xiaoli; Bromley, Robin E; Stouthamer, Richard

2012-07-01

Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence types (STs) dominated by ST11 (85%), while the remaining 22 coffee isolates defined two STs, mainly ST16 (74%). This low level of variation masked unusually large allelic differences (>1% divergence with no intermediates) at five loci (leuA, petC, malF, cysG, and holC). We developed an introgression test to detect whether these large differences were due to introgression via homologous recombination from another X. fastidiosa subspecies. Using additional sequencing around these loci, we established that the seven randomly chosen MLST targets contained seven regions of introgression totaling 2,172 bp of 4,161 bp (52%), only 409 bp (10%) of which were detected by other recombination tests. This high level of introgression suggests the hypothesis that X. fastidiosa subsp. pauca became pathogenic on citrus and coffee (crops cultivated in Brazil for several hundred years) only recently after it gained genetic variation via intersubspecific recombination, facilitating a switch from native hosts. A candidate donor is the subspecies infecting plum in the region since 1935 (possibly X. fastidiosa subsp. multiplex). This hypothesis predicts that nonrecombinant native X. fastidiosa subsp. pauca (not yet isolated) does not cause disease in citrus or coffee.

Detecting Genetic Introgression: High Levels of Intersubspecific Recombination Found in Xylella fastidiosa in Brazil

PubMed Central

Yuan, Xiaoli; Bromley, Robin E.; Stouthamer, Richard

2012-01-01

Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence types (STs) dominated by ST11 (85%), while the remaining 22 coffee isolates defined two STs, mainly ST16 (74%). This low level of variation masked unusually large allelic differences (>1% divergence with no intermediates) at five loci (leuA, petC, malF, cysG, and holC). We developed an introgression test to detect whether these large differences were due to introgression via homologous recombination from another X. fastidiosa subspecies. Using additional sequencing around these loci, we established that the seven randomly chosen MLST targets contained seven regions of introgression totaling 2,172 bp of 4,161 bp (52%), only 409 bp (10%) of which were detected by other recombination tests. This high level of introgression suggests the hypothesis that X. fastidiosa subsp. pauca became pathogenic on citrus and coffee (crops cultivated in Brazil for several hundred years) only recently after it gained genetic variation via intersubspecific recombination, facilitating a switch from native hosts. A candidate donor is the subspecies infecting plum in the region since 1935 (possibly X. fastidiosa subsp. multiplex). This hypothesis predicts that nonrecombinant native X. fastidiosa subsp. pauca (not yet isolated) does not cause disease in citrus or coffee. PMID:22544234

On the role of extracellular polymeric substances during early stages of Xylella fastidiosa biofilm formation.

PubMed

Lorite, Gabriela S; de Souza, Alessandra A; Neubauer, Daniel; Mizaikoff, Boris; Kranz, Christine; Cotta, Mônica A

2013-02-01

The structural integrity and protection of bacterial biofilms are intrinsically associated with a matrix of extracellular polymeric substances (EPS) produced by the bacteria cells. However, the role of these substances during biofilm adhesion to a surface remains largely unclear. In this study, the influence of EPS on Xylella fastidiosa biofilm formation was investigated. This bacterium is associated with economically important plant diseases; it presents a slow growth rate and thus allows us to pinpoint more precisely the early stages of cell-surface adhesion. Scanning electron microscopy and atomic force microscopy show evidence of EPS production in such early stages and around individual bacteria cells attached to the substrate surface even a few hours after inoculation. In addition, EPS formation was investigated via attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FTIR). To this end, X. fastidiosa cells were inoculated within an ATR liquid cell assembly. IR-ATR spectra clearly reveal EPS formation already during the early stages of X. fastidiosa biofilm formation, thereby providing supporting evidence for the hypothesis of the relevance of the EPS contribution to the adhesion process. Copyright © 2012 Elsevier B.V. All rights reserved.

Population structure of the bacterial pathogen Xylella fastidiosa among street trees in Washington D.C.

PubMed

Harris, Jordan Lee; Balci, Yilmaz

2015-01-01

Bacterial leaf scorch, associated with the bacterial pathogen Xylella fastidiosa, is a widely established and problematic disease of landscape ornamentals in Washington D.C. A multi-locus sequence typing analysis was performed using 10 housekeeping loci for X. fastidiosa strains in order to better understand the epidemiology of leaf scorch disease in this municipal environment. Samples were collected from 7 different tree species located throughout the District of Columbia, consisting of 101 samples of symptomatic and asymptomatic foliage from 84 different trees. Five strains of the bacteria were identified. Consistent with prior data, these strains were host specific, with only one strain associated with members of the red oak family, one strain associated with American elm, one strain associated with American sycamore, and two strains associated with mulberry. Strains found for asymptomatic foliage were the same as strains from the symptomatic foliage on individual trees. Cross transmission of the strains was not observed at sites with multiple species of infected trees within an approx. 25 m radius of one another. X. fastidiosa strain specificity observed for each genus of tree suggests a highly specialized host-pathogen relationship.

Population Structure of the Bacterial Pathogen Xylella fastidiosa among Street Trees in Washington D.C.

PubMed Central

Harris, Jordan Lee; Balci, Yilmaz

2015-01-01

Bacterial leaf scorch, associated with the bacterial pathogen Xylella fastidiosa, is a widely established and problematic disease of landscape ornamentals in Washington D.C. A multi-locus sequence typing analysis was performed using 10 housekeeping loci for X. fastidiosa strains in order to better understand the epidemiology of leaf scorch disease in this municipal environment. Samples were collected from 7 different tree species located throughout the District of Columbia, consisting of 101 samples of symptomatic and asymptomatic foliage from 84 different trees. Five strains of the bacteria were identified. Consistent with prior data, these strains were host specific, with only one strain associated with members of the red oak family, one strain associated with American elm, one strain associated with American sycamore, and two strains associated with mulberry. Strains found for asymptomatic foliage were the same as strains from the symptomatic foliage on individual trees. Cross transmission of the strains was not observed at sites with multiple species of infected trees within an approx. 25 m radius of one another. X. fastidiosa strain specificity observed for each genus of tree suggests a highly specialized host-pathogen relationship. PMID:25815838

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

PubMed

de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

2004-08-15

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

A triply cloned strain of xylella fastidiosa multiplies and induces symptoms of citrus variegated chlorosis in sweet orange

PubMed

Li; Zreik; Fernandes; Miranda; Teixeira; Ayres; Garnier; Bov

1999-08-01

Xylella fastidiosa isolate 8.1.b obtained from a sweet orange tree affected by citrus variegated chlorosis in the state of Sao Paulo, Brazil, and shown in 1993 to be the causal agent of the disease, was cloned by repeated culture in liquid and on solid PW medium, yielding triply cloned strain 9a5c. The eighth and the 16th passages of strain 9a5c were mechanically inoculated into sweet orange plants. Presence of X. fastidiosa in sweet orange leaves of shoots having grown after inoculation (first-flush shoots) was detected by DAS-ELISA and PCR. Thirty-eight days after inoculation, 70% of the 20 inoculated plants tested positive, and all plants gave strong positive reactions 90 days after inoculation. Symptoms first appeared after 3 months and were conspicuous after 5 months. X. fastidiosa was reisolated from sweet orange leaves, 44 days after inoculation. These results indicate that X. fastidiosa strain 9a5c, derived from pathogenic isolate 8.1.b by triply cloning, is also pathogenic. Strain 9a5c is now used for the X. fastidiosa genome sequencing project undertaken on a large scale in Brazil.http://link. springer-ny.com/link/service/journals/00284/bibs/39n2p106.html

Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen Xylella fastidiosa, Involved in the Synthesis of Bacterial Lipopolysaccharide.

PubMed

Alencar, Valquíria Campos; Jabes, Daniela Leite; Menegidio, Fabiano Bezerra; Sassaki, Guilherme Lanzi; de Souza, Lucas Rodrigo; Puzer, Luciano; Meneghetti, Maria Cecília Zorél; Lima, Marcelo Andrade; Tersariol, Ivarne Luis Dos Santos; de Oliveira, Regina Costa; Nunes, Luiz R

2017-02-07

Xylella fastidiosa is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce's disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to X. fastidiosa subsp. pauca, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of X. fastidiosa genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as XfUxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the X. fastidiosa uxs gene affects the LPS structure among CLS-related X. fastidiosa strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts.

Genotypic analysis of Xylella fastidiosa isolates from different hosts using sequences homologous to the Xanthomonas rpf genes.

PubMed

Meinhardt, Lyndel W; Ribeiro, Milena P M A; Coletta-Filho, Helvécio D; Dumenyo, C Korsi; Tsai, Sui M; De M Bellato, Cláudia

2003-09-01

SUMMARY This is the first report of a genotypic analysis of the phytopathogenic bacteria Xylella fastidiosa (Xf) using differences within intra- and intergenic regions of pathogenic genes. Orthologous sequences from the genome of Xf were identified for genes involved in the regulation of pathogenicity factors (rpf) from Xanthomonas campestris pv. campestris (Xcc). While the rpf genes were conserved, the chromosomal region revealed differences in gene sizes and intergenic spacings and a major translocational event when compared to Xcc. Primers were designed to amplify three regions: the intragenic region of rpfA (2354 bp), the intergenic region between rpfA and rpfB (5772 bp), and the intergenic region between rpfC and rpfF (2314 bp). Amplicons were obtained for all three regions from 32 of the 33 Xf isolates tested from citrus, grape, coffee, plum, hibiscus and periwinkle. Three Xcc isolates from cruciferous plants only generated PCR products for the rpfC-F region. Cleaved amplified polymorphic sequences (CAPS) (Taq(alpha)I) revealed differential banding profiles for the rpfA-B and rpfC-F regions. Xylella isolates were separated into seven groups via rpfA-B, of which five contained only citrus, while the other two had citrus, grape and coffee, and citrus, coffee, plum and hibiscus isolates. rpfC-F separated the isolates into three host-related groups. Citrus, coffee and hibiscus isolates formed one group, while the other two groups were comprised solely of grape and plum isolates. Xcc isolates formed an out-group. In silico analysis supports these results, which reveal the potential of the rpf genes for genotypic analysis of Xylella fastidiosa.

Whole genome evaluation of tandem repeat polymorphisms between two pathogenically similar strains of Xylella fastidiosa isolated from almond and grape in California

USDA-ARS?s Scientific Manuscript database

Whole genome tandem repeat polymorphisms were evaluated between two closely related Xylella fastidiosa strains, M23 and Temecula1, both cause almond leaf scorch disease (ALSD) and grape Pierce’s disease (PD) in California. Strain M23 was isolated from almond and the genome was sequenced in this stu...

Plant water stress effects on stylet probing behaviors of Homalodisca vitripennis (Hemiptera: Cicadellidae) associated with acquisition and inoculation of the bacterium Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter, Homalodisca vitripennis, is a xylem fluid-ingesting leafhopper that transmits Xylella fastidiosa, the causal agent of several plant diseases in the Americas. While the role of plant water stress on the population density and dispersal of H. vitripennis has been studie...

Xylella fastidiosa Differentially Accumulates Mineral Elements in Biofilm and Planktonic Cells

PubMed Central

Cobine, Paul A.; Cruz, Luisa F.; Navarrete, Fernando; Duncan, Daniel; Tygart, Melissa; De La Fuente, Leonardo

2013-01-01

Xylella fastidiosa is a bacterial plant pathogen that infects numerous plant hosts. Disease develops when the bacterium colonizes the xylem vessels and forms a biofilm. Inductively coupled plasma optical emission spectroscopy was used to examine the mineral element content of this pathogen in biofilm and planktonic states. Significant accumulations of copper (30-fold), manganese (6-fold), zinc (5-fold), calcium (2-fold) and potassium (2-fold) in the biofilm compared to planktonic cells were observed. Other mineral elements such as sodium, magnesium and iron did not significantly differ between biofilm and planktonic cells. The distribution of mineral elements in the planktonic cells loosely mirrors the media composition; however the unique mineral element distribution in biofilm suggests specific mechanisms of accumulation from the media. A cell-to-surface attachment assay shows that addition of 50 to 100 µM Cu to standard X. fastidiosa media increases biofilm, while higher concentrations (>200 µM) slow cell growth and prevent biofilm formation. Moreover cell-to-surface attachment was blocked by specific chelation of copper. Growth of X. fastidiosa in microfluidic chambers under flow conditions showed that addition of 50 µM Cu to the media accelerated attachment and aggregation, while 400 µM prevented this process. Supplementation of standard media with Mn showed increased biofilm formation and cell-to-cell attachment. In contrast, while the biofilm accumulated Zn, supplementation to the media with this element caused inhibited growth of planktonic cells and impaired biofilm formation. Collectively these data suggest roles for these minerals in attachment and biofilm formation and therefore the virulence of this pathogen. PMID:23349991

Xylella fastidiosa, a new plant pathogen that threatens global farming: Ecology, molecular biology, search for remedies.

PubMed

Bucci, Enrico M

2018-07-12

Recently, the emergence of an important alien plant pathogen in Europe was evident when the Olive Quick Decline Syndrome (OQDS), a previously unknown disease causing rapid scorching and death of the trees, invested with particular virulence a substantial portion of the vast olive wood of Southern Italy (Salento, part of the Apulia region). Early evidence indicated a connection between the OQDS and the gram-negative bacterium Xylella fastidiosa. This bacterium can target several important crops, so that researchers from all over the world have investigated its association with host plants and vectors, the molecular biology of the infection mechanism, and the molecular reaction of the infected plants. Potentially resistant or tolerant cultivars and molecular targets which might be useful to control the infection have been identified. In vitro tests of compounds active against Xylella have also been performed. In this contribution, the literature and the available data will be reviewed to provide an up-to-date picture of the currently available knowledge on the role of Xylella in OQDS, and in diseases of other plants, with focus on the emerging threats to European farming. Copyright © 2018 Elsevier Inc. All rights reserved.

The Bacterial Pathogen Xylella fastidiosa Affects the Leaf Ionome of Plant Hosts during Infection

PubMed Central

De La Fuente, Leonardo; Parker, Jennifer K.; Oliver, Jonathan E.; Granger, Shea; Brannen, Phillip M.; van Santen, Edzard; Cobine, Paul A.

2013-01-01

Xylella fastidiosa is a plant pathogenic bacterium that lives inside the host xylem vessels, where it forms biofilm believed to be responsible for disrupting the passage of water and nutrients. Here, Nicotiana tabacum was infected with X. fastidiosa, and the spatial and temporal changes in the whole-leaf ionome (i.e. the mineral and trace element composition) were measured as the host plant transitioned from healthy to diseased physiological status. The elemental composition of leaves was used as an indicator of the physiological changes in the host at a specific time and relative position during plant development. Bacterial infection was found to cause significant increases in concentrations of calcium prior to the appearance of symptoms and decreases in concentrations of phosphorous after symptoms appeared. Field-collected leaves from multiple varieties of grape, blueberry, and pecan plants grown in different locations over a four-year period in the Southeastern US showed the same alterations in Ca and P. This descriptive ionomics approach characterizes the existence of a mineral element-based response to X. fastidiosa using a model system suitable for further manipulation to uncover additional details of the role of mineral elements during plant-pathogen interactions. This is the first report on the dynamics of changes in the ionome of the host plant throughout the process of infection by a pathogen. PMID:23667547

Intersubspecific Recombination in Xylella fastidiosa Strains Native to the United States: Infection of Novel Hosts Associated with an Unsuccessful Invasion

PubMed Central

Hopkins, Donald L.; Morano, Lisa D.; Russell, Stephanie E.; Stouthamer, Richard

2014-01-01

The bacterial pathogen Xylella fastidiosa infects xylem and causes disease in many plant species in the Americas. Different subspecies of this bacterium and different genotypes within subspecies infect different plant hosts, but the genetics of host adaptation are unknown. Here we examined the hypothesis that the introduction of novel genetic variation via intersubspecific homologous recombination (IHR) facilitates host shifts. We investigated IHR in 33 X. fastidiosa subsp. multiplex isolates previously identified as recombinant based on 8 loci (7 multilocus sequence typing [MLST] loci plus 1 locus). We found significant evidence of introgression from X. fastidiosa subsp. fastidiosa in 4 of the loci and, using published data, evidence of IHR in 6 of 9 additional loci. Our data showed that IHR regions in 2 of the 4 loci were inconsistent (12 mismatches) with X. fastidiosa subsp. fastidiosa alleles found in the United States but consistent with alleles from Central America. The other two loci were consistent with alleles from both regions. We propose that the recombinant forms all originated via genomewide recombination of one X. fastidiosa subsp. multiplex ancestor with one X. fastidiosa subsp. fastidiosa donor from Central America that was introduced into the United States but subsequently disappeared. Using all of the available data, 5 plant hosts of the recombinant types were identified, 3 of which also supported non-IHR X. fastidiosa subsp. multiplex, but 2 were unique to recombinant types from blueberry (7 isolates from Georgia, 3 from Florida); and blackberry (1 each from Florida and North Carolina), strongly supporting the hypothesis that IHR facilitated a host shift to blueberry and possibly blackberry. PMID:24296499

Intersubspecific recombination in Xylella fastidiosa Strains native to the United States: infection of novel hosts associated with an unsuccessful invasion.

PubMed

Nunney, Leonard; Hopkins, Donald L; Morano, Lisa D; Russell, Stephanie E; Stouthamer, Richard

2014-02-01

The bacterial pathogen Xylella fastidiosa infects xylem and causes disease in many plant species in the Americas. Different subspecies of this bacterium and different genotypes within subspecies infect different plant hosts, but the genetics of host adaptation are unknown. Here we examined the hypothesis that the introduction of novel genetic variation via intersubspecific homologous recombination (IHR) facilitates host shifts. We investigated IHR in 33 X. fastidiosa subsp. multiplex isolates previously identified as recombinant based on 8 loci (7 multilocus sequence typing [MLST] loci plus 1 locus). We found significant evidence of introgression from X. fastidiosa subsp. fastidiosa in 4 of the loci and, using published data, evidence of IHR in 6 of 9 additional loci. Our data showed that IHR regions in 2 of the 4 loci were inconsistent (12 mismatches) with X. fastidiosa subsp. fastidiosa alleles found in the United States but consistent with alleles from Central America. The other two loci were consistent with alleles from both regions. We propose that the recombinant forms all originated via genomewide recombination of one X. fastidiosa subsp. multiplex ancestor with one X. fastidiosa subsp. fastidiosa donor from Central America that was introduced into the United States but subsequently disappeared. Using all of the available data, 5 plant hosts of the recombinant types were identified, 3 of which also supported non-IHR X. fastidiosa subsp. multiplex, but 2 were unique to recombinant types from blueberry (7 isolates from Georgia, 3 from Florida); and blackberry (1 each from Florida and North Carolina), strongly supporting the hypothesis that IHR facilitated a host shift to blueberry and possibly blackberry.

Functional characterization of replication and stability factors of an incompatibility group P-1 plasmid from Xylella fastidiosa.

PubMed

Lee, Min Woo; Rogers, Elizabeth E; Stenger, Drake C

2010-12-01

Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

A zinc, copper and citric acid biocomplex shows promise for control of Xylella fastidiosa subsp. pauca in olive trees in Apulia region (southern Italy)

USDA-ARS?s Scientific Manuscript database

The bacterium Xylella fastidiosa subsp. pauca is associated with the “olive quick decline syndrome” in the Apulia region of southern Italy. To investigate control of this phytopathogen, a compound containing zinc and copper complexed with citric-acid hydracids (Dentamet®) was evaluated for in vitro ...

Lipopolysaccharide O-antigen delays plant innate immune recognition of Xylella fastidiosa.

PubMed

Rapicavoli, Jeannette N; Blanco-Ulate, Barbara; Muszyński, Artur; Figueroa-Balderas, Rosa; Morales-Cruz, Abraham; Azadi, Parastoo; Dobruchowska, Justyna M; Castro, Claudia; Cantu, Dario; Roper, M Caroline

2018-01-26

Lipopolysaccharides (LPS) are among the known pathogen-associated molecular patterns (PAMPs). LPSs are potent elicitors of PAMP-triggered immunity (PTI), and bacteria have evolved intricate mechanisms to dampen PTI. Here we demonstrate that Xylella fastidiosa (Xf), a hemibiotrophic plant pathogenic bacterium, possesses a long chain O-antigen that enables it to delay initial plant recognition, thereby allowing it to effectively skirt initial elicitation of innate immunity and establish itself in the host. Lack of the O-antigen modifies plant perception of Xf and enables elicitation of hallmarks of PTI, such as ROS production specifically in the plant xylem tissue compartment, a tissue not traditionally considered a spatial location of PTI. To explore translational applications of our findings, we demonstrate that pre-treatment of plants with Xf LPS primes grapevine defenses to confer tolerance to Xf challenge.

Specific Detection and Identification of American Mulberry-Infecting and Italian Olive-Associated Strains of Xylella fastidiosa by Polymerase Chain Reaction

PubMed Central

Guan, Wei; Shao, Jonathan; Elbeaino, Toufic; Davis, Robert E.; Zhao, Tingchang; Huang, Qi

2015-01-01

Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains. PMID:26061051

Specific Detection and Identification of American Mulberry-Infecting and Italian Olive-Associated Strains of Xylella fastidiosa by Polymerase Chain Reaction.

PubMed

Guan, Wei; Shao, Jonathan; Elbeaino, Toufic; Davis, Robert E; Zhao, Tingchang; Huang, Qi

2015-01-01

Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains.

The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation.

PubMed

Merfa, Marcus V; Niza, Bárbara; Takita, Marco A; De Souza, Alessandra A

2016-01-01

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions.

The MqsRA Toxin-Antitoxin System from Xylella fastidiosa Plays a Key Role in Bacterial Fitness, Pathogenicity, and Persister Cell Formation

PubMed Central

Merfa, Marcus V.; Niza, Bárbara; Takita, Marco A.; De Souza, Alessandra A.

2016-01-01

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis—CVC) activated by copper stress, and has been described in Escherichia coli as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X. fastidiosa and reveal new insights into the physiology of phytopathogen-host interactions. PMID:27375608

Seasonal Fluctuations of Sap-Feeding Insect Species Infected by Xylella fastidiosa in Apulian Olive Groves of Southern Italy.

PubMed

Ben Moussa, Issam Eddine; Mazzoni, Valerio; Valentini, Franco; Yaseen, Thaer; Lorusso, Donato; Speranza, Stefano; Digiaro, Michele; Varvaro, Leonardo; Krugner, Rodrigo; D'Onghia, Anna Maria

2016-08-01

A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Large-scale intersubspecific recombination in the plant-pathogenic bacterium Xylella fastidiosa is associated with the host shift to mulberry.

PubMed

Nunney, Leonard; Schuenzel, Erin L; Scally, Mark; Bromley, Robin E; Stouthamer, Richard

2014-05-01

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.

Large-Scale Intersubspecific Recombination in the Plant-Pathogenic Bacterium Xylella fastidiosa Is Associated with the Host Shift to Mulberry

PubMed Central

Schuenzel, Erin L.; Scally, Mark; Bromley, Robin E.; Stouthamer, Richard

2014-01-01

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry. PMID:24610840

Production of Xylella fastidiosa diffusible signal factor in transgenic grape causes pathogen confusion and reduction in severity of Pierce's disease.

PubMed

Lindow, Steven; Newman, Karyn; Chatterjee, Subhadeep; Baccari, Clelia; Lavarone, Anthony T; Ionescu, Michael

2014-03-01

The rpfF gene from Xylella fastidiosa, encoding the synthase for diffusible signal factor (DSF), was expressed in 'Freedom' grape to reduce the pathogen's growth and mobility within the plant. Symptoms in such plants were restricted to near the point of inoculation and incidence of disease was two- to fivefold lower than in the parental line. Both the longitudinal and lateral movement of X. fastidiosa in the xylem was also much lower. DSF was detected in both leaves and xylem sap of RpfF-expressing plants using biological sensors, and both 2-Z-tetradecenoic acid, previously identified as a component of X. fastidiosa DSF, and cis-11-methyl-2-dodecenoic acid were detected in xylem sap using electrospray ionization mass spectrometry. A higher proportion of X. fastidiosa cells adhered to xylem vessels of the RpfF-expressing line than parental 'Freedom' plants, reflecting a higher adhesiveness of the pathogen in the presence of DSF. Disease incidence in RpfF-expressing plants in field trials in which plants were either mechanically inoculated with X. fastidiosa or subjected to natural inoculation by sharpshooter vectors was two- to fourfold lower in than that of the parental line. The number of symptomatic leaves on infected shoots was reduced proportionally more than the incidence of infection, reflecting a decreased ability of X. fastidiosa to move within DSF-producing plants.

Tolerance to oxidative stress is required for maximal xylem colonization by the xylem-limited bacterial phytopathogen, Xylella fastidiosa.

PubMed

Wang, Peng; Lee, Yunho; Igo, Michele M; Roper, M Caroline

2017-09-01

Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem-limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H 2 O 2 ) relative to the wild-type. In addition, during early stages of grapevine infection, the survival rate was 1000-fold lower for the oxyR mutant than for the wild-type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell-cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

PubMed Central

2010-01-01

Background Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. Conclusions Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon. PMID:20799976

A cell–cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa

PubMed Central

Chatterjee, Subhadeep; Wistrom, Christina; Lindow, Steven E.

2008-01-01

Cell–cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF. PMID:18268318

A cell-cell signaling sensor is required for virulence and insect transmission of Xylella fastidiosa.

PubMed

Chatterjee, Subhadeep; Wistrom, Christina; Lindow, Steven E

2008-02-19

Cell-cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF.

Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

PubMed

Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

2015-10-01

Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

O Antigen Modulates Insect Vector Acquisition of the Bacterial Plant Pathogen Xylella fastidiosa

PubMed Central

Rapicavoli, Jeannette N.; Kinsinger, Nichola; Perring, Thomas M.; Backus, Elaine A.; Shugart, Holly J.; Walker, Sharon

2015-01-01

Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. PMID:26386068

Analysis of gene expression in two growth states of Xylella fastidiosa and its relationship with pathogenicity.

PubMed

de Souza, Alessandra A; Takita, Marco A; Coletta-Filho, Helvécio D; Caldana, Camila; Goldman, Gustavo H; Yanai, Giane M; Muto, Nair H; de Oliveira, Regina C; Nunes, Luiz R; Machado, Marcos A

2003-10-01

Xylella fastidiosa is a plant pathogen responsible for diseases of economically important crops. Although there is considerable disagreement about its mechanism of pathogenicity, blockage of the vessels is one of the most accepted hypotheses. Loss of virulence by this bacterium was observed after serial passages in axenic culture. To confirm the loss of pathogenicity of X. fastidiosa, the causing agent of citrus variegated chlorosis (CVC), freshly-isolated bacteria (first passage [FP] condition) as well as bacteria obtained after 46 passages in axenic culture (several passage [SP] condition) were inoculated into sweet orange and periwinkle plants. Using real time quantitative polymerase chain reaction, we verified that the colonization of FP cells was more efficient for both hosts. The sequence of the complete X. fastidiosa genome allowed the construction of a DNA microarray that was used to investigate the total changes in gene expression associated with the FP condition. Most genes found to be induced in the FP condition were associated with adhesion and probably with adaptation to the host environment. This report represents the first study of the transcriptome of this pathogen, which has recently gained more importance, since the genome of several strains has been either partially or entirely sequenced.

Water deficit modulates the response of Vitis vinifera to the Pierce's disease pathogen Xylella fastidiosa.

PubMed

Choi, Hong-Kyu; Iandolino, Alberto; da Silva, Francisco Goes; Cook, Douglas R

2013-06-01

Pierce's disease, caused by the bacterium Xylella fastidiosa, is one of the most devastating diseases of cultivated grape, currently restricted to the Americas. To test the long-standing hypothesis that Pierce's disease results from pathogen-induced drought stress, we used the Affymetrix Vitis GeneChip to compare the transcriptional response of Vitis vinifera to Xylella infection, water deficit, or a combination of the two stresses. The results reveal a redirection of gene transcription involving 822 genes with a minimum twofold change (P < 0.05), including the upregulation of transcripts for phenylpropanoid and flavonoid biosynthesis, pathogenesis-related proteins, abscisic acid- and jasmonic acid-responsive biosynthesis, and downregulation of transcripts related to photosynthesis, growth, and nutrition. Although the transcriptional response of plants to Xylella infection was largely distinct from the response of healthy plants to water stress, we find that 138 of the pathogen-induced genes exhibited a significantly stronger transcriptional response when plants were simultaneously exposed to infection and drought stress, suggesting a strong interaction between disease and water deficit. This interaction between drought stress and disease was mirrored in planta at the physiological level for aspects of water relations and photosynthesis and in terms of the severity of disease symptoms and the extent of pathogen colonization, providing a molecular correlate of the classical concept of the disease triangle in which environment impacts disease severity.

The exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence, and vector transmission.

PubMed

Killiny, N; Martinez, R Hernandez; Dumenyo, C Korsi; Cooksey, D A; Almeida, R P P

2013-09-01

Exopolysaccharides (EPS) synthesized by plant-pathogenic bacteria are generally essential for virulence. The role of EPS produced by the vector-transmitted bacterium Xylella fastidiosa was investigated by knocking out two genes implicated in the EPS biosynthesis, gumD and gumH. Mutant strains were affected in growth characteristics in vitro, including adhesion to surfaces and biofilm formation. In addition, different assays were used to demonstrate that the mutant strains produced significantly less EPS compared with the wild type. Furthermore, gas chromatography-mass spectrometry showed that both mutant strains did not produce oligosaccharides. Biologically, the mutants were deficient in movement within plants, resulting in an avirulent phenotype. Additionally, mutant strains were affected in transmission by insects: they were very poorly transmitted by and retained within vectors. The gene expression profile indicated upregulation of genes implicated in cell-to-cell signaling and adhesins while downregulation in genes was required for within-plant movement in EPS-deficient strains. These results suggest an essential role for EPS in X. fastidiosa interactions with both plants and insects.

Preliminary findings suggest that vector feeding behaviors controlling inoculation of Xylella fastidiosa are performed less on Vitis arizonica than on V. vinifera ‘Chardonnay’

USDA-ARS?s Scientific Manuscript database

The most successful example of classical grapevine breeding for resistance to Xylella fastidiosa (Xf) is the PdR1 gene, which mediates resistance to Xf multiplication and spread in the host, once Xf has been inoculated. No effort has been made to determine whether resistance of PdR1 or its parent wi...

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-08-01

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

Grape Cultivar and Sap Culture Conditions Affect the Development of Xylella fastidiosa Phenotypes Associated with Pierce's Disease.

PubMed

Hao, Lingyun; Zaini, Paulo A; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2016-01-01

Xylella fastidiosa is a xylem-limited bacterium in plant hosts and causes Pierce's disease (PD) of grapevines, which differ in susceptibility according to the Vitis species (spp.). In this work we compared X. fastidiosa biofilm formation and population dynamics when cultured in xylem saps from PD-susceptible and -resistant Vitis spp. under different conditions. Behaviors in a closed-culture system were compared to those in different sap-renewal cultures that would more closely mimic the physicochemical environment encountered in planta. Significant differences in biofilm formation and growth in saps from PD-susceptible and -resistant spp. were only observed using sap renewal culture. Compared to saps from susceptible V. vinifera, those from PD-resistant V. aestivalis supported lower titers of X. fastidiosa and less biofilm and V. champinii suppressed both growth and biofilm formation, behaviors which are correlated with disease susceptibility. Furthermore, in microfluidic chambers X. fastidiosa formed thick mature biofilm with three-dimensional (3-D) structures, such as pillars and mounds, in saps from all susceptible spp. In contrast, only small aggregates of various shapes were formed in saps from four out of five of the resistant spp.; sap from the resistant spp. V. mustangensis was an exception in that it also supported thick lawns of biofilm but not the above described 3-D structures typically seen in a mature biofilm from the susceptible saps. Our findings provide not only critical technical information for future bioassays, but also suggest further understanding of PD susceptibility.

Grape Cultivar and Sap Culture Conditions Affect the Development of Xylella fastidiosa Phenotypes Associated with Pierce's Disease

PubMed Central

Hoch, Harvey C.; Burr, Thomas J.; Mowery, Patricia

2016-01-01

Xylella fastidiosa is a xylem-limited bacterium in plant hosts and causes Pierce’s disease (PD) of grapevines, which differ in susceptibility according to the Vitis species (spp.). In this work we compared X. fastidiosa biofilm formation and population dynamics when cultured in xylem saps from PD-susceptible and -resistant Vitis spp. under different conditions. Behaviors in a closed-culture system were compared to those in different sap-renewal cultures that would more closely mimic the physicochemical environment encountered in planta. Significant differences in biofilm formation and growth in saps from PD-susceptible and -resistant spp. were only observed using sap renewal culture. Compared to saps from susceptible V. vinifera, those from PD-resistant V. aestivalis supported lower titers of X. fastidiosa and less biofilm and V. champinii suppressed both growth and biofilm formation, behaviors which are correlated with disease susceptibility. Furthermore, in microfluidic chambers X. fastidiosa formed thick mature biofilm with three-dimensional (3-D) structures, such as pillars and mounds, in saps from all susceptible spp. In contrast, only small aggregates of various shapes were formed in saps from four out of five of the resistant spp.; sap from the resistant spp. V. mustangensis was an exception in that it also supported thick lawns of biofilm but not the above described 3-D structures typically seen in a mature biofilm from the susceptible saps. Our findings provide not only critical technical information for future bioassays, but also suggest further understanding of PD susceptibility. PMID:27508296

Feeding Behaviors That Promote Inoculation Of Xylella fastidiosa Are Performed Less By Glassy-winged Sharpshooters on Resistant Vitis Candicans Than On Susceptible V. vinifera cv. ‘Chardonnay’

USDA-ARS?s Scientific Manuscript database

Development of grape varieties resistant to Pierce’s Disease, caused by the lethal bacterium Xylella fastidiosa (Xf), is considered the most sustainable, long-term solution to the disease. Grape breeders are working to develop varieties resistant to multiplication and spread of the bacterium, by in...

Seasonal Abundance and Natural Inoculativity of Insect Vectors of Xylella fastidiosa in Oklahoma Tree Nurseries and Vineyards.

PubMed

Overall, Lisa M; Rebek, Eric J

2015-12-01

Xylella fastidiosa is the causative agent of diseases of perennial plants including peach, plum, elm, oak, pecan, and grape. This bacterial pathogen is transmitted by xylem-feeding insects. In recent years, Pierce's disease of grape has been detected in 10 counties in central and northeastern Oklahoma, prompting further investigation of the disease epidemiology in this state. We surveyed vineyards and tree nurseries in Oklahoma for potential insect vectors to determine species composition, infectivity, and natural inoculativity of commonly captured insect vectors. Yellow sticky cards were used to sample insect fauna at each location. Insects were removed from sticky cards and screened for X. fastidiosa using immunocapture-PCR to determine their infectivity. A second objective was to test the natural inoculativity of insect vectors that are found in vineyards. Graphocephala versuta (Say), Graphocephala coccinea (Forster), Paraulacizes irrorata (F.), Oncometopia orbona (F.), Cuerna costalis (F.), and Entylia carinata Germar were collected from vineyards and taken back to the lab to determine their natural inoculativity. Immunocapture-PCR was used to test plant and insect samples for presence of X. fastidiosa. The three most frequently captured species from vineyards and tree nurseries were G. versuta, Clastoptera xanthocephala Germar, and O. orbona. Of those insects screened for X. fastidiosa, 2.4% tested positive for the bacterium. Field-collected G. versuta were inoculative to both ragweed and alfalfa. Following a 7-d inoculation access period, a higher percentage of alfalfa became infected than ragweed. Results from this study provide insight into the epidemiology of X. fastidiosa in Oklahoma. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Real-time investigation of mannosyltransferase function of a Xylella fastidiosa recombinant GumH protein using QCM-D.

PubMed

Alves, Claudia A; Pedroso, Mariele M; de Moraes, Marcela C; Souza, Dulce H F; Cass, Quezia B; Faria, Ronaldo C

2011-05-20

Xylella fastidiosa is a gram-negative bacterium that causes serious diseases in economically important crops, including grapevine, coffee, and citrus fruits. X. fastidiosa colonizes the xylem vessels of the infected plants, thereby blocking water and nutrient transport. The genome sequence of X. fastidiosa has revealed an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide (EPS) named fastidian gum that can be related with the pathogenicity of this bacterium. The α-1,3-mannosyltransferase (GumH) enzyme from X. fastidiosa is involved in fastidian gum production. GumH is responsible for the transfer of mannose from guanosine diphosphate mannose (GDP-man) to the cellobiose-pyrophosphate-polyprenol carrier lipid (CPP-Lip) during the assembly and biosynthesis of EPS. In this work, a method for real-time detection of recombinant GumH enzymatic activity was successfully developed using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). The QCM-D transducer was strategically modified with CPP-Lip by using a solid-supported lipid bilayer that makes use of a self-assembled monolayer of 1-undecanethiol. Monitoring the real-time CPP-Lip QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The real-time QCM-D determination of mannosyltransferase function was validated by a High Performance Liquid Chromatography (LC) method developed for determination of GDP produced by enzymatic reaction. LC results confirmed the activity of recombinant GumH protein, which is the first enzyme involved in the biosynthesis of the EPS from X. fastidiosa enzymatically characterized. Copyright © 2011 Elsevier Inc. All rights reserved.

Differential gene expression in Xylella fastidiosa 9a5c during co-cultivation with the endophytic bacterium Methylobacterium mesophilicum SR1.6/6.

PubMed

Dourado, Manuella Nóbrega; Santos, Daiene Souza; Nunes, Luiz Roberto; Costa de Oliveira, Regina Lúcia Batista da; de Oliveira, Marcus Vinicius; Araújo, Welington Luiz

2015-12-01

Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC), colonizes plant xylem, reducing sap flow, and inducing internerval chlorosis, leaf size reduction, necrosis, and harder and smaller fruits. This bacterium may be transmitted from plant to plant by sharpshooter insects, including Bucephalogonia xanthopis. The citrus endophytic bacterium Methylobacterium mesophilicum SR1.6/6 colonizes citrus xylem and previous studies showed that this strain is also transferred from plant to plant by B. xanthopis (Insecta), suggesting that this endophytic bacterium may interact with X. fastidiosa in planta and inside the insect vector during co-transmission by the same insect vector. To better understand the X. fastidiosa behavior in the presence of M. mesophilicum, we evaluated the X. fastidiosa transcriptional profile during in vitro interaction with M. mesophilicum SR1.6/6. The results showed that during co-cultivation, X. fastidiosa down-regulated genes related to growth and up-regulated genes related to energy production, stress, transport, and motility, suggesting the existence of a specific adaptive response to the presence of M. mesophilicum in the culture medium. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell Wall-Degrading Enzymes Enlarge the Pore Size of Intervessel Pit Membranes in Healthy and Xylella fastidiosa-Infected Grapevines1[C][W][OA

PubMed Central

Pérez-Donoso, Alonso G.; Sun, Qiang; Roper, M. Caroline; Greve, L. Carl; Kirkpatrick, Bruce; Labavitch, John M.

2010-01-01

The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4- β -glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce's disease. PMID:20107028

O antigen modulates insect vector acquisition of the bacterial plant pathogen Xylella fastidiosa.

PubMed

Rapicavoli, Jeannette N; Kinsinger, Nichola; Perring, Thomas M; Backus, Elaine A; Shugart, Holly J; Walker, Sharon; Roper, M Caroline

2015-12-01

Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Calcium transcriptionally regulates the biofilm machinery of Xylella fastidiosa to promote continued biofilm development in batch cultures.

PubMed

Parker, Jennifer K; Chen, Hongyu; McCarty, Sara E; Liu, Lawrence Y; De La Fuente, Leonardo

2016-05-01

The functions of calcium (Ca) in bacteria are less characterized than in eukaryotes, where its role has been studied extensively. The plant-pathogenic bacterium Xylella fastidiosa has several virulence features that are enhanced by increased Ca concentrations, including biofilm formation. However, the specific mechanisms driving modulation of this feature are unclear. Characterization of biofilm formation over time showed that 4 mM Ca supplementation produced denser biofilms that were still developing at 96 h, while biofilm in non-supplemented media had reached the dispersal stage by 72 h. To identify changes in global gene expression in X. fastidiosa grown in supplemental Ca, RNA-Seq of batch culture biofilm cells was conducted at three 24-h time intervals. Results indicate that a variety of genes are differentially expressed in response to Ca, including genes related to attachment, motility, exopolysaccharide synthesis, biofilm formation, peptidoglycan synthesis, regulatory functions, iron homeostasis, and phages. Collectively, results demonstrate that Ca supplementation induces a transcriptional response that promotes continued biofilm development, while biofilm cells in nonsupplemented media are driven towards dispersion of cells from the biofilm structure. These results have important implications for disease progression in planta, where xylem sap is the source of Ca and other nutrients for X. fastidiosa. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Zinc Detoxification Is Required for Full Virulence and Modification of the Host Leaf Ionome by Xylella fastidiosa.

PubMed

Navarrete, Fernando; De La Fuente, Leonardo

2015-04-01

Zinc (Zn) is an essential element for all forms of life because it is a structural or catalytic cofactor of many proteins, but it can have toxic effects at high concentrations; thus, microorganisms must tightly regulate its levels. Here, we evaluated the role of Zn homeostasis proteins in the virulence of the xylem-limited bacterium Xylella fastidiosa, causal agent of Pierce's disease of grapevine, among other diseases. Two mutants of X. fastidiosa 'Temecula' affected in genes which regulate Zn homeostasis (zur) and Zn detoxification (czcD) were constructed. Both knockouts showed increased sensitivity to Zn at physiologically relevant concentrations and increased intracellular accumulation of this metal compared with the wild type. Increased Zn sensitivity was correlated with decreased growth in grapevine xylem sap, reduced twitching motility, and downregulation of exopolysaccharide biosynthetic genes. Tobacco plants inoculated with either knockout mutant showed reduced foliar symptoms and a much reduced (czcD) or absent (zur) modification of the leaf ionome (i.e., the mineral nutrient and trace element composition), as well as reduced bacterial populations. The results show that detoxification of Zn is crucial for the virulence of X. fastidiosa and verifies our previous findings that modification of the host leaf ionome correlates with bacterial virulence.

Proteome analysis of the plant pathogen Xylella fastidiosa reveals major cellular and extracellular proteins and a peculiar codon bias distribution.

PubMed

Smolka, Marcus Bustamante; Martins-de-Souza, Daniel; Martins, Daniel; Winck, Flavia Vischi; Santoro, Carlos Eduardo; Castellari, Rafael Ramos; Ferrari, Fernanda; Brum, Itaraju Junior; Galembeck, Eduardo; Della Coletta Filho, Helvécio; Machado, Marcos Antonio; Marangoni, Sergio; Novello, Jose Camillo

2003-02-01

The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis. Although its complete genome is already sequenced, X. fastidiosa is very poorly characterized by biochemical approaches at the protein level. In an initial effort to characterize protein expression in X. fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c. Of particular interest for the study of pathogenesis are adhesion and secreted proteins. Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels. Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins. These data suggest that the intercellular space of X. fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis. A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X. fastidiosa. A database of the X. fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).

Spatial Genetic Structure of Coffee-Associated Xylella fastidiosa Populations Indicates that Cross Infection Does Not Occur with Sympatric Citrus Orchards.

PubMed

Francisco, Carolina S; Ceresini, Paulo C; Almeida, Rodrigo P P; Coletta-Filho, Helvécio D

2017-04-01

Xylella fastidiosa, an economically important plant-pathogenic bacterium, infects both coffee and citrus trees in Brazil. Although X. fastidiosa in citrus is well studied, knowledge about the population structure of this bacterium infecting coffee remains unknown. Here, we studied the population structure of X. fastidiosa infecting coffee trees in São Paulo State, Brazil, in four regions where citrus is also widely cultivated. Genotyping of over 500 isolates from coffee plants using 14 genomic microsatellite markers indicated that populations were largely geographically isolated, as previously found with populations of X. fastidiosa infecting citrus. These results were supported by a clustering analysis, which indicated three major genetic groups among the four sampled regions. Overall, approximately 38% of isolates showed significant membership coefficients not related to their original geographical populations (i.e., migrants), characterizing a significant degree of genotype flow among populations. To determine whether admixture occurred between isolates infecting citrus and coffee plants, one site with citrus and coffee orchards adjacent to each other was selected; over 100 isolates were typed from each host plant. No signal of natural admixture between citrus- and coffee-infecting isolates was found; artificial cross-infection assays with representative isolates also yielded no successful cross infection. A comparison determined that X. fastidiosa populations from coffee have higher genetic diversity and allelic richness compared with citrus. The results showed that coffee and citrus X. fastidiosa populations are effectively isolated from each other and, although coffee populations are spatially structured, migration has an important role in shaping diversity.

DNA microarray-based genome comparison of a pathogenic and a nonpathogenic strain of Xylella fastidiosa delineates genes important for bacterial virulence.

PubMed

Koide, Tie; Zaini, Paulo A; Moreira, Leandro M; Vêncio, Ricardo Z N; Matsukuma, Adriana Y; Durham, Alan M; Teixeira, Diva C; El-Dorry, Hamza; Monteiro, Patrícia B; da Silva, Ana Claudia R; Verjovski-Almeida, Sergio; da Silva, Aline M; Gomes, Suely L

2004-08-01

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

DNA Microarray-Based Genome Comparison of a Pathogenic and a Nonpathogenic Strain of Xylella fastidiosa Delineates Genes Important for Bacterial Virulence†

PubMed Central

Koide, Tie; Zaini, Paulo A.; Moreira, Leandro M.; Vêncio, Ricardo Z. N.; Matsukuma, Adriana Y.; Durham, Alan M.; Teixeira, Diva C.; El-Dorry, Hamza; Monteiro, Patrícia B.; da Silva, Ana Claudia R.; Verjovski-Almeida, Sergio; da Silva, Aline M.; Gomes, Suely L.

2004-01-01

Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease. PMID:15292146

Initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa related to the human cytosolic 5'-nucleotidase I.

PubMed

Santos, Clelton A; Saraiva, Antonio M; Toledo, Marcelo A S; Beloti, Lilian L; Crucello, Aline; Favaro, Marianna T P; Horta, Maria A C; Santiago, André S; Mendes, Juliano S; Souza, Alessandra A; Souza, Anete P

2013-01-01

The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Genome-Wide Analysis Provides Evidence on the Genetic Relatedness of the Emergent Xylella fastidiosa Genotype in Italy to Isolates from Central America.

PubMed

Giampetruzzi, Annalisa; Saponari, Maria; Loconsole, Giuliana; Boscia, Donato; Savino, Vito Nicola; Almeida, Rodrigo P P; Zicca, Stefania; Landa, Blanca B; Chacón-Diaz, Carlos; Saldarelli, Pasquale

2017-07-01

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp

Comparative analyses of Xanthomonas and Xylella complete genomes.

PubMed

Moreira, Leandro M; De Souza, Robson F; Digiampietri, Luciano A; Da Silva, Ana C R; Setubal, João C

2005-01-01

Computational analyses of four bacterial genomes of the Xanthomonadaceae family reveal new unique genes that may be involved in adaptation, pathogenicity, and host specificity. The Xanthomonas genus presents 3636 unique genes distributed in 1470 families, while Xylella genus presents 1026 unique genes distributed in 375 families. Among Xanthomonas-specific genes, we highlight a large number of cell wall degrading enzymes, proteases, and iron receptors, a set of energy metabolism genes, second copy of the type II secretion system, type III secretion system, flagella and chemotactic machinery, and the xanthomonadin synthesis gene cluster. Important genes unique to the Xylella genus are an additional copy of a type IV pili gene cluster and the complete machinery of colicin V synthesis and secretion. Intersections of gene sets from both genera reveal a cluster of genes homologous to Salmonella's SPI-7 island in Xanthomonas axonopodis pv citri and Xylella fastidiosa 9a5c, which might be involved in host specificity. Each genome also presents important unique genes, such as an HMS cluster, the kdgT gene, and O-antigen in Xanthomonas axonopodis pv citri; a number of avrBS genes and a distinct O-antigen in Xanthomonas campestris pv campestris, a type I restriction-modification system and a nickase gene in Xylella fastidiosa 9a5c, and a type II restriction-modification system and two genes related to peptidoglycan biosynthesis in Xylella fastidiosa temecula 1. All these differences imply a considerable number of gene gains and losses during the divergence of the four lineages, and are associated with structural genome modifications that may have a direct relation with the mode of transmission, adaptation to specific environments and pathogenicity of each organism.

Characterization of the Xylella fastidiosa PD1671 gene encoding degenerate c-di-GMP GGDEF/EAL domains, and its role in the development of Pierce's disease.

PubMed

Cursino, Luciana; Athinuwat, Dusit; Patel, Kelly R; Galvani, Cheryl D; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

2015-01-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Living in two worlds: the plant and insect lifestyles of Xylella fastidiosa.

PubMed

Chatterjee, Subhadeep; Almeida, Rodrigo P P; Lindow, Steven

2008-01-01

Diseases caused by Xylella fastidiosa have attained great importance worldwide as the pathogen and its insect vectors have been disseminated. Since this is the first plant pathogenic bacterium for which a complete genome sequence was determined, much progress has been made in understanding the process by which it spreads within the xylem vessels of susceptible plants as well as the traits that contribute to its acquisition and transmission by sharpshooter vectors. Although this pathogen shares many similarities with Xanthomonas species, such as its use of a small fatty acid signal molecule to coordinate virulence gene expression, the traits that it utilizes to cause disease and the manner in which they are regulated differ substantially from those of related plant pathogens. Its complex lifestyle as both a plant and insect colonist involves traits that are in conflict with these stages, thus apparently necessitating the use of a gene regulatory scheme that allows cells expressing different traits to co-occur in the plant.

Whole-genome comparative analysis of three phytopathogenic Xylella fastidiosa strains.

PubMed

Bhattacharyya, Anamitra; Stilwagen, Stephanie; Ivanova, Natalia; D'Souza, Mark; Bernal, Axel; Lykidis, Athanasios; Kapatral, Vinayak; Anderson, Iain; Larsen, Niels; Los, Tamara; Reznik, Gary; Selkov, Eugene; Walunas, Theresa L; Feil, Helene; Feil, William S; Purcell, Alexander; Lassez, Jean-Louis; Hawkins, Trevor L; Haselkorn, Robert; Overbeek, Ross; Predki, Paul F; Kyrpides, Nikos C

2002-09-17

Xylella fastidiosa (Xf) causes wilt disease in plants and is responsible for major economic and crop losses globally. Owing to the public importance of this phytopathogen we embarked on a comparative analysis of the complete genome of Xf pv citrus and the partial genomes of two recently sequenced strains of this species: Xf pv almond and Xf pv oleander, which cause leaf scorch in almond and oleander plants, respectively. We report a reanalysis of the previously sequenced Xf 9a5c (CVC, citrus) strain and the two "gapped" Xf genomes revealing ORFs encoding critical functions in pathogenicity and conjugative transfer. Second, a detailed whole-genome functional comparison was based on the three sequenced Xf strains, identifying the unique genes present in each strain, in addition to those shared between strains. Third, an "in silico" cellular reconstruction of these organisms was made, based on a comparison of their core functional subsystems that led to a characterization of their conjugative transfer machinery, identification of potential differences in their adhesion mechanisms, and highlighting of the absence of a classical quorum-sensing mechanism. This study demonstrates the effectiveness of comparative analysis strategies in the interpretation of genomes that are closely related.

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

PubMed

Li, Wenbin; Teixeira, Diva C; Hartung, John S; Huang, Qi; Duan, Yongping; Zhou, Lijuan; Chen, Jianchi; Lin, Hong; Lopes, Silvio; Ayres, A Juliano; Levy, Laurene

2013-01-01

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the

Response of Xylella fastidiosa to zinc: decreased culturability, increased exopolysaccharide production, and formation of resilient biofilms under flow conditions.

PubMed

Navarrete, Fernando; De La Fuente, Leonardo

2014-02-01

The bacterial plant pathogen Xylella fastidiosa produces biofilm that accumulates in the host xylem vessels, affecting disease development in various crops and bacterial acquisition by insect vectors. Biofilms are sensitive to the chemical composition of the environment, and mineral elements being transported in the xylem are of special interest for this pathosystem. Here, X. fastidiosa liquid cultures were supplemented with zinc and compared with nonamended cultures to determine the effects of Zn on growth, biofilm, and exopolysaccharide (EPS) production under batch and flow culture conditions. The results show that Zn reduces growth and biofilm production under both conditions. However, in microfluidic chambers under liquid flow and with constant bacterial supplementation (closer to conditions inside the host), a dramatic increase in biofilm aggregates was seen in the Zn-amended medium. Biofilms formed under these conditions were strongly attached to surfaces and were not removed by medium flow. This phenomenon was correlated with increased EPS production in stationary-phase cells grown under high Zn concentrations. Zn did not cause greater adhesion to surfaces by individual cells. Additionally, viability analyses suggest that X. fastidiosa may be able to enter the viable but nonculturable state in vitro, and Zn can hasten the onset of this state. Together, these findings suggest that Zn can act as a stress factor with pleiotropic effects on X. fastidiosa and indicate that, although Zn could be used as a bactericide treatment, it could trigger the undesired effect of stronger biofilm formation upon reinoculation events.

Response of Xylella fastidiosa to Zinc: Decreased Culturability, Increased Exopolysaccharide Production, and Formation of Resilient Biofilms under Flow Conditions

PubMed Central

Navarrete, Fernando

2014-01-01

The bacterial plant pathogen Xylella fastidiosa produces biofilm that accumulates in the host xylem vessels, affecting disease development in various crops and bacterial acquisition by insect vectors. Biofilms are sensitive to the chemical composition of the environment, and mineral elements being transported in the xylem are of special interest for this pathosystem. Here, X. fastidiosa liquid cultures were supplemented with zinc and compared with nonamended cultures to determine the effects of Zn on growth, biofilm, and exopolysaccharide (EPS) production under batch and flow culture conditions. The results show that Zn reduces growth and biofilm production under both conditions. However, in microfluidic chambers under liquid flow and with constant bacterial supplementation (closer to conditions inside the host), a dramatic increase in biofilm aggregates was seen in the Zn-amended medium. Biofilms formed under these conditions were strongly attached to surfaces and were not removed by medium flow. This phenomenon was correlated with increased EPS production in stationary-phase cells grown under high Zn concentrations. Zn did not cause greater adhesion to surfaces by individual cells. Additionally, viability analyses suggest that X. fastidiosa may be able to enter the viable but nonculturable state in vitro, and Zn can hasten the onset of this state. Together, these findings suggest that Zn can act as a stress factor with pleiotropic effects on X. fastidiosa and indicate that, although Zn could be used as a bactericide treatment, it could trigger the undesired effect of stronger biofilm formation upon reinoculation events. PMID:24271184

Cell-to-cell signaling in Xylella fastidiosa suppresses movement and xylem vessel colonization in grape.

PubMed

Chatterjee, Subhadeep; Newman, Karyn L; Lindow, Steven E

2008-10-01

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein-marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and

Identification of a response regulator involved in surface attachment, cell-cell aggregation, exopolysaccharide production and virulence in the plant pathogen Xylella fastidiosa.

PubMed

Voegel, Tanja M; Doddapaneni, Harshavardhan; Cheng, Davis W; Lin, Hong; Stenger, Drake C; Kirkpatrick, Bruce C; Roper, M Caroline

2013-04-01

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

PubMed

Rosselli-Murai, Luciana K; Sforça, Maurício L; Sassonia, Rogério C; Azzoni, Adriano R; Murai, Marcelo J; de Souza, Anete P; Zeri, Ana C

2012-10-01

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex. Most of the residues involved in contacts to DNA are located around the first and second loops and in the first helix at a positively charged side of the protein surface. The overall structure of the Xylella H-NS C-terminal domain differ significantly from Escherichia coli and Salmonella enterica H-NS proteins, even though the DNA binding motif in loop 2 adopt similar conformation, as well as β-strand 2 and loop 1. Interestingly, we have also found that the DNA binding site is expanded to include helix 1, which is not seen in the other structures. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of the Xylella fastidiosa PD1671 Gene Encoding Degenerate c-di-GMP GGDEF/EAL Domains, and Its Role in the Development of Pierce’s Disease

PubMed Central

Cursino, Luciana; Athinuwat, Dusit; Patel, Kelly R.; Galvani, Cheryl D.; Zaini, Paulo A.; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C.; Burr, Thomas J.; Mowery, Patricia

2015-01-01

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce’s disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence. PMID:25811864

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

PubMed

Warren, Jeremy G; Lincoln, James E; Kirkpatrick, Bruce C

2015-01-01

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras

PubMed Central

Warren, Jeremy G.; Lincoln, James E.; Kirkpatrick, Bruce C.

2015-01-01

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

Xylella taiwanensis sp. nov. cause of pear leaf scorch disease in Taiwan

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa is a group of xylem-limited and nutritionally fastidious plant pathogenic bacteria. While mostly found in the Americas, new X. fastidiosa strains have been reported from other continents such as Asia, including a pear leaf scorch (PLS) strain from Taiwan. Current taxonomy of X. fa...

Xylella genomics and bacterial pathogenicity to plants.

PubMed

Dow, J M; Daniels, M J

2000-12-01

Xylella fastidiosa, a pathogen of citrus, is the first plant pathogenic bacterium for which the complete genome sequence has been published. Inspection of the sequence reveals high relatedness to many genes of other pathogens, notably Xanthomonas campestris. Based on this, we suggest that Xylella possesses certain easily testable properties that contribute to pathogenicity. We also present some general considerations for deriving information on pathogenicity from bacterial genomics. Copyright 2000 John Wiley & Sons, Ltd.

A rhamnose-rich O-antigen mediates adhesion, virulence, and host colonization for the xylem-limited phytopathogen Xylella fastidiosa.

PubMed

Clifford, Jennifer C; Rapicavoli, Jeannette N; Roper, M Caroline

2013-06-01

Xylella fastidiosa is a gram-negative, xylem-limited bacterium that causes a lethal disease of grapevine called Pierce's disease. Lipopolysaccharide (LPS) composes approximately 75% of the outer membrane of gram-negative bacteria and, because it is largely displayed on the cell surface, it mediates interactions between the bacterial cell and its surrounding environment. LPS is composed of a conserved lipid A-core oligosaccharide component and a variable O-antigen portion. By targeting a key O-antigen biosynthetic gene, we demonstrate the contribution of the rhamnose-rich O-antigen to surface attachment, cell-cell aggregation, and biofilm maturation: critical steps for successful infection of the host xylem tissue. Moreover, we have demonstrated that a fully formed O-antigen moiety is an important virulence factor for Pierce's disease development in grape and that depletion of the O-antigen compromises its ability to colonize the host. It has long been speculated that cell-surface polysaccharides play a role in X. fastidiosa virulence and this study confirms that LPS is a major virulence factor for this important agricultural pathogen.

Ribosome Display of Combinatorial Antibody Libraries Derived from Mice Immunized with Heat-Killed Xylella fastidiosa and the Selection of MopB-Specific Single-Chain Antibodies

PubMed Central

Azizi, Armaghan; Arora, Arinder; Markiv, Anatoliy; Lampe, David J.; Miller, Thomas A.

2012-01-01

Pierce's disease is a devastating lethal disease of Vitus vinifera grapevines caused by the bacterium Xylella fastidiosa. There is no cure for Pierce's disease, and control is achieved predominantly by suppressing transmission of the glassy-winged sharpshooter insect vector. We present a simple robust approach for the generation of panels of recombinant single-chain antibodies against the surface-exposed elements of X. fastidiosa that may have potential use in diagnosis and/or disease transmission blocking studies. In vitro combinatorial antibody ribosome display libraries were assembled from immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed X. fastidiosa. The libraries were used in a single round of selection against an outer membrane protein, MopB, resulting in the isolation of a panel of recombinant antibodies. The potential use of selected anti-MopB antibodies was demonstrated by the successful application of the 4XfMopB3 antibody in an enzyme-linked immunosorbent assay (ELISA), a Western blot assay, and an immunofluorescence assay (IFA). These immortalized in vitro recombinant single-chain antibody libraries generated against heat-killed X. fastidiosa are a resource for the Pierce's disease research community that may be readily accessed for the isolation of antibodies against a plethora of X. fastidiosa surface-exposed antigenic molecules. PMID:22327580

Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility.

PubMed

Cruz, Luisa F; Cobine, Paul A; De La Fuente, Leonardo

2012-03-01

Xylella fastidiosa is a plant-pathogenic bacterium that forms biofilms inside xylem vessels, a process thought to be influenced by the chemical composition of xylem sap. In this work, the effect of calcium on the production of X. fastidiosa biofilm and movement was analyzed under in vitro conditions. After a dose-response study with 96-well plates using eight metals, the strongest increase of biofilm formation was observed when medium was supplemented with at least 1.0 mM CaCl(2). The removal of Ca by extracellular (EGTA, 1.5 mM) and intracellular [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), 75 μM] chelators reduced biofilm formation without compromising planktonic growth. The concentration of Ca influenced the force of adhesion to the substrate, biofilm thickness, cell-to-cell aggregation, and twitching motility, as shown by assays with microfluidic chambers and other assays. The effect of Ca on attachment was lost when cells were treated with tetracycline, suggesting that Ca has a metabolic or regulatory role in cell adhesion. A double mutant (fimA pilO) lacking type I and type IV pili did not improve biofilm formation or attachment when Ca was added to the medium, while single mutants of type I (fimA) or type IV (pilB) pili formed more biofilm under conditions of higher Ca concentrations. The concentration of Ca in the medium did not significantly influence the levels of exopolysaccharide produced. Our findings indicate that the role of Ca in biofilm formation may be related to the initial surface and cell-to-cell attachment and colonization stages of biofilm establishment, which rely on critical functions by fimbrial structures.

Calcium Increases Xylella fastidiosa Surface Attachment, Biofilm Formation, and Twitching Motility

PubMed Central

Cruz, Luisa F.; Cobine, Paul A.

2012-01-01

Xylella fastidiosa is a plant-pathogenic bacterium that forms biofilms inside xylem vessels, a process thought to be influenced by the chemical composition of xylem sap. In this work, the effect of calcium on the production of X. fastidiosa biofilm and movement was analyzed under in vitro conditions. After a dose-response study with 96-well plates using eight metals, the strongest increase of biofilm formation was observed when medium was supplemented with at least 1.0 mM CaCl2. The removal of Ca by extracellular (EGTA, 1.5 mM) and intracellular [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM), 75 μM] chelators reduced biofilm formation without compromising planktonic growth. The concentration of Ca influenced the force of adhesion to the substrate, biofilm thickness, cell-to-cell aggregation, and twitching motility, as shown by assays with microfluidic chambers and other assays. The effect of Ca on attachment was lost when cells were treated with tetracycline, suggesting that Ca has a metabolic or regulatory role in cell adhesion. A double mutant (fimA pilO) lacking type I and type IV pili did not improve biofilm formation or attachment when Ca was added to the medium, while single mutants of type I (fimA) or type IV (pilB) pili formed more biofilm under conditions of higher Ca concentrations. The concentration of Ca in the medium did not significantly influence the levels of exopolysaccharide produced. Our findings indicate that the role of Ca in biofilm formation may be related to the initial surface and cell-to-cell attachment and colonization stages of biofilm establishment, which rely on critical functions by fimbrial structures. PMID:22194297

Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa.

PubMed

Catani, Cleide Ferreira; Azzoni, Adriano Rodrigues; Paula, Débora Pires; Tada, Susely Ferraz Siqueira; Rosselli, Luciana Kauer; de Souza, Anete Pereira; Yano, Tomomasa

2004-10-01

In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37 degrees C, with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy.

Nanofilms of hyaluronan/chitosan assembled layer-by-layer: An antibacterial surface for Xylella fastidiosa.

PubMed

Hernández-Montelongo, Jacobo; Nascimento, Vicente F; Murillo, Duber; Taketa, Thiago B; Sahoo, Prasana; de Souza, Alessandra A; Beppu, Marisa M; Cotta, Monica A

2016-01-20

In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum. Copyright © 2015 Elsevier Ltd. All rights reserved.

Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation.

PubMed

Janissen, Richard; Murillo, Duber M; Niza, Barbara; Sahoo, Prasana K; Nobrega, Marcelo M; Cesar, Carlos L; Temperini, Marcia L A; Carvalho, Hernandes F; de Souza, Alessandra A; Cotta, Monica A

2015-04-20

Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.

Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation

PubMed Central

Janissen, Richard; Murillo, Duber M.; Niza, Barbara; Sahoo, Prasana K.; Nobrega, Marcelo M.; Cesar, Carlos L.; Temperini, Marcia L. A.; Carvalho, Hernandes F.; de Souza, Alessandra A.; Cotta, Monica A.

2015-01-01

Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation. PMID:25891045

Surface physicochemical properties at the micro and nano length scales: role on bacterial adhesion and Xylella fastidiosa biofilm development.

PubMed

Lorite, Gabriela S; Janissen, Richard; Clerici, João H; Rodrigues, Carolina M; Tomaz, Juarez P; Mizaikoff, Boris; Kranz, Christine; de Souza, Alessandra A; Cotta, Mônica A

2013-01-01

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.

Surface Physicochemical Properties at the Micro and Nano Length Scales: Role on Bacterial Adhesion and Xylella fastidiosa Biofilm Development

PubMed Central

Lorite, Gabriela S.; Janissen, Richard; Clerici, João H.; Rodrigues, Carolina M.; Tomaz, Juarez P.; Mizaikoff, Boris; Kranz, Christine; de Souza, Alessandra A.; Cotta, Mônica A.

2013-01-01

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant. PMID:24073256

Acquisition of Xyllela fastidiosa causes changes to the inoculation behavior (EPG X wave) of an efficient sharpshooter vector

USDA-ARS?s Scientific Manuscript database

Xylella fastidiosa (Xf) is a foregut-borne bacterium that is inoculated into xylem cells of a healthy plant during feeding by sharpshooter vectors. Inoculation occurs during salivation and egestion behaviors that are likely represented by the sharpshooter X wave. The objective of this study was to t...

Factors affecting the initial adhesion and retention of the plant pathogen Xylella fastidiosa in the foregut of an insect vector.

PubMed

Killiny, Nabil; Almeida, Rodrigo P P

2014-01-01

Vector transmission of bacterial plant pathogens involves three steps: pathogen acquisition from an infected host, retention within the vector, and inoculation of cells into susceptible tissue of an uninfected plant. In this study, a combination of plant and artificial diet systems were used to determine the importance of several genes on the initial adhesion and retention of the bacterium Xylella fastidiosa to an efficient insect vector. Mutant strains included fimbrial (fimA and pilB) and afimbrial (hxfA and hxfB) adhesins and three loci involved in regulatory systems (rpfF, rpfC, and cgsA). Transmission assays with variable retention time indicated that HxfA and HxfB were primarily important for early adhesion to vectors, while FimA was necessary for both adhesion and retention. The long pilus protein PilB was not deficient in initial adhesion but may be important for retention. Genes upregulated under the control of rpfF are important for both initial adhesion and retention, as transmission rates of this mutant strain were initially low and decreased over time, while disruption of rpfC and cgsA yielded trends similar to that shown by the wild-type control. Because induction of an X. fastidiosa transmissible state requires pectin, a series of experiments were used to test the roles of a polygalacturonase (pglA) and the pectin and galacturonic acid carbohydrates on the transmission of X. fastidiosa. Results show that galacturonic acid, or PglA activity breaking pectin into its major subunit (galacturonic acid), is required for X. fastidiosa vector transmission using an artificial diet system. This study shows that early adhesion and retention of X. fastidiosa are mediated by different factors. It also illustrates that the interpretation of results of vector transmission experiments, in the context of vector-pathogen interaction studies, is highly dependent on experimental design.

Factors Affecting the Initial Adhesion and Retention of the Plant Pathogen Xylella fastidiosa in the Foregut of an Insect Vector

PubMed Central

Almeida, Rodrigo P. P.

2014-01-01

Vector transmission of bacterial plant pathogens involves three steps: pathogen acquisition from an infected host, retention within the vector, and inoculation of cells into susceptible tissue of an uninfected plant. In this study, a combination of plant and artificial diet systems were used to determine the importance of several genes on the initial adhesion and retention of the bacterium Xylella fastidiosa to an efficient insect vector. Mutant strains included fimbrial (fimA and pilB) and afimbrial (hxfA and hxfB) adhesins and three loci involved in regulatory systems (rpfF, rpfC, and cgsA). Transmission assays with variable retention time indicated that HxfA and HxfB were primarily important for early adhesion to vectors, while FimA was necessary for both adhesion and retention. The long pilus protein PilB was not deficient in initial adhesion but may be important for retention. Genes upregulated under the control of rpfF are important for both initial adhesion and retention, as transmission rates of this mutant strain were initially low and decreased over time, while disruption of rpfC and cgsA yielded trends similar to that shown by the wild-type control. Because induction of an X. fastidiosa transmissible state requires pectin, a series of experiments were used to test the roles of a polygalacturonase (pglA) and the pectin and galacturonic acid carbohydrates on the transmission of X. fastidiosa. Results show that galacturonic acid, or PglA activity breaking pectin into its major subunit (galacturonic acid), is required for X. fastidiosa vector transmission using an artificial diet system. This study shows that early adhesion and retention of X. fastidiosa are mediated by different factors. It also illustrates that the interpretation of results of vector transmission experiments, in the context of vector-pathogen interaction studies, is highly dependent on experimental design. PMID:24185853

Plant water stress effects on stylet probing behaviors of Homalodisca vitripennis (Hemiptera: Cicadellidae) associated with acquisition and inoculation of the bacterium Xylella fastidiosa.

PubMed

Krugner, Rodrigo; Backus, Elaine A

2014-02-01

ABSTRACT The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), is a xylem fluid-ingesting leafhopper that transmits Xylella fastidiosa Wells et al., a plant-infecting bacterium that causes several plant diseases in the Americas. Although the role of plant water stress on the population density and dispersal ofH. vitripennis has been studied, nothing is known about the effects of plant water stress on the transmission of X. fastidiosa by H. vitripennis. A laboratory study was conducted to determine the influence of plant water stress on the sharpshooter stylet probing behaviors associated with the acquisition and inoculation of X. fastidiosa. Electrical penetration graph was used to monitor H. vitripennis feeding behaviors for 20-h periods on citrus [Citrus sinensis (L.) Osbeck] and almond [Prunus dulcis (Miller) D.A. Webb] plants subjected to levels of water stress. Adult H. vitripennis successfully located xylem vessels, then performed behaviors related to the evaluation of the xylem cell and fluid, and finally ingested xylem fluid from citrus and almond plants under the tested fluid tensions ranging from -5.5 to -33.0 bars and -6.0 to -24.5 bars, respectively. In general, long and frequent feeding events associated with the acquisition and inoculation of X. fastidiosa were observed only in fully irrigated plants (i.e., >-10 bars), which suggests that even low levels of plant water stress may reduce the spread of X. fastidiosa. Results provided insights to disease epidemiology and support the hypothesis that application of regulated deficit irrigation has the potential to reduce the incidence of diseases caused by X.fastidiosa by reducing the number of vectors and by decreasing pathogen transmission efficiency.

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro.

PubMed

Santos, Clelton A; Toledo, Marcelo A S; Trivella, Daniela B B; Beloti, Lilian L; Schneider, Dilaine R S; Saraiva, Antonio M; Crucello, Aline; Azzoni, Adriano R; Souza, Alessandra A; Aparicio, Ricardo; Souza, Anete P

2012-10-01

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. © 2012 The Authors Journal compilation © 2012 FEBS.

Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa.

PubMed

Backus, Elaine A; Andrews, Kim B; Shugart, Holly J; Carl Greve, L; Labavitch, John M; Alhaddad, Hasan

2012-07-01

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insect's salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away

A new member of the aldo-keto reductase family from the plant pathogen Xylella fastidiosa.

PubMed

Rosselli, Luciana K; Oliveira, Cristiano L P; Azzoni, Adriano R; Tada, Susely F S; Catani, Cleide F; Saraiva, Antonio M; Soares, José Sérgio M; Medrano, Francisco J; Torriani, Iris L; Souza, Anete P

2006-09-15

The Xylella fastidiosa genome program generated a large number of gene sequences that belong to pathogenicity, virulence and adaptation categories from this important plant pathogen. One of these genes (XF1729) encodes a protein similar to a superfamily of aldo-keto reductase together with a number of structurally and functionally related NADPH-dependent oxidoreductases. In this work, the similar sequence XF1729 from X. fastidiosa was cloned onto the pET32Xa/LIC vector in order to overexpress a recombinant His-tag fusion protein in Escherichia coli BL21(DE3). The expressed protein in the soluble fraction was purified by immobilized metal affinity chromatography (agarose-IDA-Ni resin). Secondary structure contents were verified by circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) measurements furnish general structural parameters and provide a strong indication that the protein has a monomeric form in solution. Also, ab initio calculations show that the protein has some similarities with a previously crystallized aldo-keto reductase protein. The recombinant XF1729 purified to homogeneity catalyzed the reduction of dl-glyceraldehyde (K(cat) 2.26s(-1), Km 8.20+/-0.98 mM) and 2-nitrobenzaldehyde (K(cat) 11.74 s(-1), Km 0.14+/-0.04 mM) in the presence of NADPH. The amino acid sequence deduced from XF1729 showed the highest identity (40% or higher) with several functional unknown proteins. Among the identified AKRs, we found approximately 29% of identity with YakC (AKR13), 30 and 28% with AKR11A and AKR11B, respectively. The results establish XF1729 as the new member of AKR family, AKR13B1. Finally, the first characterization by gel filtration chromatography assays indicates that the protein has an elongated shape, which generates an apparent higher molecular weight. The study of this protein is an effort to fight X. fastidiosa, which causes tremendous losses in many economically important plants.

RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response.

PubMed

Rodrigues, Carolina M; de Souza, Alessandra A; Takita, Marco A; Kishi, Luciano T; Machado, Marcos A

2013-10-03

Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is one the most important citrus diseases, and affects all varieties of sweet orange (Citrus sinensis L. Osb). On the other hand, among the Citrus genus there are different sources of resistance against X. fastidiosa. For these species identifying these defense genes could be an important step towards obtaining sweet orange resistant varieties through breeding or genetic engineering. To assess these genes we made use of mandarin (C. reticulata Blanco) that is known to be resistant to CVC and shares agronomical characteristics with sweet orange. Thus, we investigated the gene expression in Ponkan mandarin at one day after infection with X. fastidiosa, using RNA-seq. A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange. Gene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later. Among the induced transcripts, we identified genes encoding proteins similar to Pattern Recognition Receptors. Furthermore, many genes involved in secondary metabolism, biosynthesis and cell wall modification were upregulated as well as in synthesis of abscisic acid, jasmonic acid and auxin. This work demonstrated that the defense response to the perception of bacteria involves cell wall modification and activation of hormone pathways, which probably lead to the induction of other defense-related genes. We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen.

RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response

PubMed Central

2013-01-01

Background Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is one the most important citrus diseases, and affects all varieties of sweet orange (Citrus sinensis L. Osb). On the other hand, among the Citrus genus there are different sources of resistance against X. fastidiosa. For these species identifying these defense genes could be an important step towards obtaining sweet orange resistant varieties through breeding or genetic engineering. To assess these genes we made use of mandarin (C. reticulata Blanco) that is known to be resistant to CVC and shares agronomical characteristics with sweet orange. Thus, we investigated the gene expression in Ponkan mandarin at one day after infection with X. fastidiosa, using RNA-seq. A set of genes considered key elements in the resistance was used to confirm its regulation in mandarin compared with the susceptible sweet orange. Results Gene expression analysis of mock inoculated and infected tissues of Ponkan mandarin identified 667 transcripts repressed and 724 significantly induced in the later. Among the induced transcripts, we identified genes encoding proteins similar to Pattern Recognition Receptors. Furthermore, many genes involved in secondary metabolism, biosynthesis and cell wall modification were upregulated as well as in synthesis of abscisic acid, jasmonic acid and auxin. Conclusions This work demonstrated that the defense response to the perception of bacteria involves cell wall modification and activation of hormone pathways, which probably lead to the induction of other defense-related genes. We also hypothesized the induction of auxin-related genes indicates that resistant plants initially recognize X. fastidiosa as a necrotrophic pathogen. PMID:24090429

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

PubMed

Caserta, R; Souza-Neto, R R; Takita, M A; Lindow, S E; De Souza, A A

2017-11-01

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU.

PubMed

Shi, Xiang Yang; Dumenyo, C Korsi; Hernandez-Martinez, Rufina; Azad, Hamid; Cooksey, Donald A

2007-11-01

Many virulence genes in plant bacterial pathogens are coordinately regulated by "global" regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.

Specific Fluorescence in Situ Hybridization (FISH) Test to Highlight Colonization of Xylem Vessels by Xylella fastidiosa in Naturally Infected Olive Trees (Olea europaea L.)

PubMed Central

Cardinale, Massimiliano; Luvisi, Andrea; Meyer, Joana B.; Sabella, Erika; De Bellis, Luigi; Cruz, Albert C.; Ampatzidis, Yiannis; Cherubini, Paolo

2018-01-01

The colonization behavior of the Xylella fastidiosa strain CoDiRO, the causal agent of olive quick decline syndrome (OQDS), within the xylem of Olea europaea L. is still quite controversial. As previous literature suggests, even if xylem vessel occlusions in naturally infected olive plants were observed, cell aggregation in the formation of occlusions had a minimal role. This observation left some open questions about the whole behavior of the CoDiRO strain and its actual role in OQDS pathogenesis. In order to evaluate the extent of bacterial infection in olive trees and the role of bacterial aggregates in vessel occlusions, we tested a specific fluorescence in situ hybridization (FISH) probe (KO 210) for X. fastidiosa and quantified the level of infection and vessel occlusion in both petioles and branches of naturally infected and non-infected olive trees. All symptomatic petioles showed colonization by X. fastidiosa, especially in the larger innermost vessels. In several cases, the vessels appeared completely occluded by a biofilm containing bacterial cells and extracellular matrix and the frequent colonization of adjacent vessels suggested a horizontal movement of the bacteria. Infected symptomatic trees had 21.6 ± 10.7% of petiole vessels colonized by the pathogen, indicating an irregular distribution in olive tree xylem. Thus, our observations point out the primary role of the pathogen in olive vessel occlusions. Furthermore, our findings indicate that the KO 210 FISH probe is suitable for the specific detection of X. fastidiosa. PMID:29681910

The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines

USDA-ARS?s Scientific Manuscript database

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant and complementary strain contai...

Global expression profile of biofilm resistance to antimicrobial compounds in the plant-pathogenic bacterium Xylella fastidiosa reveals evidence of persister cells.

PubMed

Muranaka, Lígia S; Takita, Marco A; Olivato, Jacqueline C; Kishi, Luciano T; de Souza, Alessandra A

2012-09-01

Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable.

Global Expression Profile of Biofilm Resistance to Antimicrobial Compounds in the Plant-Pathogenic Bacterium Xylella fastidiosa Reveals Evidence of Persister Cells

PubMed Central

Muranaka, Lígia S.; Takita, Marco A.; Olivato, Jacqueline C.; Kishi, Luciano T.

2012-01-01

Investigations of biofilm resistance response rarely focus on plant-pathogenic bacteria. Since Xylella fastidiosa is a multihost plant-pathogenic bacterium that forms biofilm in the xylem, the behavior of its biofilm in response to antimicrobial compounds needs to be better investigated. We analyzed here the transcriptional profile of X. fastidiosa subsp. pauca in response to inhibitory and subinhibitory concentrations of copper and tetracycline. Copper-based products are routinely used to control citrus diseases in the field, while antibiotics are more widely used for bacterial control in mammals. The use of antimicrobial compounds triggers specific responses to each compound, such as biofilm formation and phage activity for copper. Common changes in expression responses comprise the repression of genes associated with metabolic functions and movement and the induction of toxin-antitoxin systems, which have been associated with the formation of persister cells. Our results also show that these cells were found in the population at a ca. 0.05% density under inhibitory conditions for both antimicrobial compounds and that pretreatment with subinhibitory concentration of copper increases this number. No previous report has detected the presence of these cells in X. fastidiosa population, suggesting that this could lead to a multidrug tolerance response in the biofilm under a stressed environment. This is a mechanism that has recently become the focus of studies on resistance of human-pathogenic bacteria to antibiotics and, based on our data, it seems to be more broadly applicable. PMID:22730126

[Survey of sharpshooters (Hemiptera: Cicadellidae) associated with Xylella fastidiosa transmission in citrus groves of the North Coast of Bahia State].

PubMed

De Miranda, Marcelo P; Lopes, João R S; Do Nascimento, Antonio S; Dos Santos, José L; Cavichioli, Rodney R

2009-01-01

The causal agent of citrus variegated clorosis, Xylella fastidiosa, is transmitted by leafhoppers of the subfamily Cicadellinae, whose species vary regionally. The goal of this study was to identify potential vectors of this pathogen in citrus groves of Bahia North Coast, Brazil. The survey was done from March/2002 to February/2003 in three seven- to nine-year-old sweet orange (Citrus sinensis, Pêra variety) groves located in Rio Real, BA. Fifteen yellow sticky cards (8.5x11.5 cm) were installed 40 m apart in each grove, hanged at 1.5 m high on the upper north side of citrus canopies, and replaced fortnightly. A sweep net was periodically used to sample leafhoppers on herbaceous weeds inside the groves, by selecting five points at random and performing 30 sweeps in each point. A total of 1,360 specimens of 49 Auchenorrhyncha species were collected in this study, mostly in the family Cicadellidae (90.2%). The subfamily Cicadellinae, which includes the sharpshooter vectors of X. fastidiosa, showed the largest number of species (14) and specimens (84.8%). Acrogonia flagellata Young, A. citrina Marucci & Cavichioli, Homalodisca spottii Takiya, Cavichioli & McKamey and an unidentified Cicadellini (species 1) were the dominant species trapped on citrus canopies, while Hortensia similis (Walker) and Erythrogonia dubia (Medler) were dominant in the weedy vegetation. Among the Cicadellinae species already known as vectors of X. fastidiosa in citrus, only A. citrina, Bucephalogonia xanthophis (Berg) e Ferrariana trivittata (Signoret) were found. The two latter species were accidentally trapped by sweep net in the weedy vegetation.

Citrus Functional Genomics and Molecular Modeling in Relation to Citrus sinensis (Sweet Orange) Infection with Xylella fastidiosa (Citrus Variegated Chlorosis).

PubMed

Dwivedi, Upendra N; Tiwari, Sameeksha; Prasanna, Pragya; Awasthi, Manika; Singh, Swati; Pandey, Veda P

2016-08-01

Citrus are among the economically most important fruit tree crops in the world. Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa infection, is a serious disease limiting citrus production at a global scale. With availability of citrus genomic resources, it is now possible to compare citrus expressed sequence tag (EST) data sets and identify single-nucleotide polymorphisms (SNPs) within and among different citrus cultivars that can be exploited for citrus resistance to infections, citrus breeding, among others. We report here, for the first time, SNPs in the EST data sets of X. fastidiosa-infected Citrus sinensis (sweet orange) and their functional annotation that revealed the involvement of eight C. sinensis candidate genes in CVC pathogenesis. Among these genes were xyloglucan endotransglycosylase, myo-inositol-1-phosphate synthase, and peroxidase were found to be involved in plant cell wall metabolism. These have been further investigated by molecular modeling for their role in CVC infection and defense. Molecular docking analyses of the wild and the mutant (SNP containing) types of the selected three enzymes with their respective substrates revealed a significant decrease in the binding affinity of substrates for the mutant enzymes, thus suggesting a decrease in the catalytic efficiency of these enzymes during infection, thereby facilitating a favorable condition for infection by the pathogen. These findings offer novel agrigenomics insights in developing future molecular targets and strategies for citrus fruit cultivation in ways that are resistant to X. fastidiosa infection, and by extension, with greater harvesting efficiency and economic value.

Characterization of an oxidative stress response regulator, homologous to Escherichia coli OxyR, from the phytopathogen Xylella fastidiosa.

PubMed

Toledo, M A S; Schneider, D R; Azzoni, A R; Favaro, M T P; Pelloso, A C; Santos, C A; Saraiva, A M; Souza, A P

2011-02-01

The OxyR oxidative stress transcriptional regulator is a DNA-binding protein that belongs to the LysR-type transcriptional regulators (LTTR) family. It has the ability to sense oxidative species inside the cell and to trigger the cell's response, activating the transcription of genes involved in scavenging oxidative species. In the present study, we have overexpressed, purified and characterized the predicted OxyR homologue (orf xf1273) of the phytopathogen Xylella fastidiosa. This bacterium is the causal agent of citrus variegated chlorosis (CVC) disease caused by the 9a5c strain, resulting in economic and social losses. The secondary structure of the recombinant protein was analyzed by circular dichroism. Gel filtration showed that XfoxyR is a dimer in solution. Gel shift assays indicated that it does bind to its own predicted promoter under in vitro conditions. However, considering our control experiment we cannot state that this interaction occurs in vivo. Functional complementation assays indicated that xfoxyR is able to restore the oxidative stress response in an oxyr knockout Escherichia coli strain. These results show that the predicted orfxf1273 codes for a transcriptional regulator, homologous to E. coli OxyR, involved in the oxidative stress response. This may be important for X. fastidiosa to overcome the defense mechanisms of its host during the infection and colonization processes. Copyright © 2010 Elsevier Inc. All rights reserved.

Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

PubMed

Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

2015-10-01

The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. Copyright © 2015 Elsevier B.V. All rights reserved.

Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain Stag's Leap.

PubMed

Chen, J; Wu, F; Zheng, Z; Deng, X; Burbank, L P; Stenger, D C

2016-04-21

ITALIC! Xylella fastidiosasubsp. ITALIC! fastidiosacauses Pierce's disease of grapevine. Presented here is the draft genome sequence of the Stag's Leap strain, previously used in pathogenicity/virulence assays to evaluate grapevine germplasm bearing Pierce's disease resistance and a phenotypic assessment of knockout mutants to determine gene function. Copyright © 2016 Chen et al.

Characterization of a putative Xylella fastidiosa diffusible signal factor by HRGC-EI-MS.

PubMed

Colnaghi Simionato, Ana Valéria; da Silva, Denise Santos; Lambais, Marcio Rodrigues; Carrilho, Emanuel

2007-10-01

Xylella fastidiosa (X.f.) is a plant pathogen with high levels of genomic similarity to Xanthomonas campestris pv. campestris (X.c.c.). It has been shown that X. fastidiosa synthesizes a putative diffusible signal factor (X.f.-DSF) that activates regulation of pathogenicity factor (rpf) genes in a X.c.c. reporter system, which might be involved in the regulation of pathogenesis associated genes as in X.c.c., as well as in quorum-sensing. The nature of the X.f.-DSF is not known, whereas the X.c.c.-DSF has been identified as cis-11-methyl-2-dodecenoic acid. In this work, the chemical nature of a putative X.f.-DSF molecule, able to restore endoglucanase activity in a X.c.c. rpfF mutant, was investigated as if it was a fatty acid derivative. Bioassays with X.c.c. reporter bacterium and X.f. culture extracts, based on endoglucanase restoration activity, were also carried out in order to confirm the DSFs molecules similarities. For this reason, a gas chromatography-mass spectrometry method was developed with standard fatty acids methyl esters mixtures. The retention time, as well as the fragmentation patterns, of each standard was used to identify the DSF molecule synthesized by X.f. in the culture medium. Typical ester fragmentation patterns (the derivatized analyte) were observed, such as: McLafferty rearrangement and migration of the Hdelta followed by 1,4-hydrogen shift and cleavage of the bond Cbeta--Cgamma, confirming the nature of this molecule. This confirmation was corroborated by the common peaks in both spectra. Besides, the observed retention time reinforces our conclusion since it corresponds to a methyl ester with 15 carbons. Since the X.f.-DSF molecule was tentatively identified as 12-methyl-tetradecanoic acid (by mass spectra library comparison), this standard compound was also analyzed, strongly suggesting that this is the identification of such a molecule. To our knowledge, this is the first time a DSF produced by X.f. has been characterized.

Characterization of a putative Xylella fastidiosa diffusible signal factor by HRGC-EI-MS.

PubMed

Colnaghi Simionato, Ana Valéria; da Silva, Denise Santos; Lambais, Marcio Rodrigues; Carrilho, Emanuel

2007-04-01

Xylella fastidiosa (X.f.) is a plant pathogen with high levels of genomic similarity to Xanthomonas campestris pv. campestris (X.c.c.). It has been shown that X. fastidiosa synthesizes a putative diffusible signal factor (X.f.-DSF) that activates regulation of pathogenicity factor (rpf) genes in a X.c.c. reporter system, which might be involved in the regulation of pathogenesis associated genes as in X.c.c., as well as in quorum-sensing. The nature of the X.f.-DSF is not known, whereas the X.c.c.-DSF has been identified as cis-11-methyl-2-dodecenoic acid. In this work, the chemical nature of a putative X.f.-DSF molecule, able to restore endoglucanase activity in a X.c.c. rpfF mutant, was investigated as if it was a fatty acid derivative. Bioassays with X.c.c. reporter bacterium and X.f. culture extracts, based on endoglucanase restoration activity, were also carried out in order to confirm the DSFs molecules similarities. For this reason, a gas chromatography-mass spectrometry method was developed with standard fatty acids methyl esters mixtures. The retention time, as well as the fragmentation patterns, of each standard was used to identify the DSF molecule synthesized by X.f. in the culture medium. Typical ester fragmentation patterns (the derivatized analyte) were observed, such as: McLafferty rearrangement and migration of the Hdelta followed by 1,4-hydrogen shift and cleavage of the bond Cbeta-Cgamma, confirming the nature of this molecule. This confirmation was corroborated by the common peaks in both spectra. Besides, the observed retention time reinforces our conclusion since it corresponds to a methyl ester with 15 carbons. Since the X.f.-DSF molecule was tentatively identified as 12-methyl-tetradecanoic acid (by mass spectra library comparison), this standard compound was also analyzed, strongly suggesting that this is the identification of such a molecule. To our knowledge, this is the first time a DSF produced by X.f. has been characterized. Copyright

N-acetylcysteine in agriculture, a novel use for an old molecule: focus on controlling the plant-pathogen Xylella fastidiosa.

PubMed

Muranaka, Lígia S; Giorgiano, Thais E; Takita, Marco A; Forim, Moacir R; Silva, Luis F C; Coletta-Filho, Helvécio D; Machado, Marcos A; de Souza, Alessandra A

2013-01-01

Xylella fastidiosa is a plant pathogen bacterium that causes diseases in many different crops. In citrus, it causes Citrus Variegated Chlorosis (CVC). The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants, and there is not yet any method to directly reduce populations of this pathogen in the field. In this study, we investigated the inhibitory effect of N-Acetylcysteine (NAC), a cysteine analogue used mainly to treat human diseases, on X. fastidiosa in different experimental conditions. Concentrations of NAC over 1 mg/mL reduced bacterial adhesion to glass surfaces, biofilm formation and the amount of exopolysaccharides (EPS). The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to X. fastidiosa-infected plants in hydroponics, fertigation, and adsorbed to organic fertilizer (NAC-Fertilizer). HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL but not at 6 mg/mL. Sweet orange plants with CVC symptoms treated with NAC (0.48 and 2.4 mg/mL) in hydroponics showed clear symptom remission and reduction in bacterial population, as analyzed by quantitative PCR and bacterial isolation. Experiments using fertigation and NAC-Fertilizer were done to simulate a condition closer to that normally is used in the field. For both, significant symptom remission and a reduced bacterial growth rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment increased to around eight months. This is the first report of the anti-bacterial effect of NAC against a phytopathogenic bacterium. The results obtained in this work together with the characteristics of this molecule indicate that the use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogenic bacteria.

N-Acetylcysteine in Agriculture, a Novel Use for an Old Molecule: Focus on Controlling the Plant–Pathogen Xylella fastidiosa

PubMed Central

Muranaka, Lígia S.; Giorgiano, Thais E.; Takita, Marco A.; Forim, Moacir R.; Silva, Luis F. C.; Coletta-Filho, Helvécio D.; Machado, Marcos A.; de Souza, Alessandra A.

2013-01-01

Xylella fastidiosa is a plant pathogen bacterium that causes diseases in many different crops. In citrus, it causes Citrus Variegated Chlorosis (CVC). The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants, and there is not yet any method to directly reduce populations of this pathogen in the field. In this study, we investigated the inhibitory effect of N-Acetylcysteine (NAC), a cysteine analogue used mainly to treat human diseases, on X. fastidiosa in different experimental conditions. Concentrations of NAC over 1 mg/mL reduced bacterial adhesion to glass surfaces, biofilm formation and the amount of exopolysaccharides (EPS). The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to X. fastidiosa-infected plants in hydroponics, fertigation, and adsorbed to organic fertilizer (NAC-Fertilizer). HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL but not at 6 mg/mL. Sweet orange plants with CVC symptoms treated with NAC (0.48 and 2.4 mg/mL) in hydroponics showed clear symptom remission and reduction in bacterial population, as analyzed by quantitative PCR and bacterial isolation. Experiments using fertigation and NAC-Fertilizer were done to simulate a condition closer to that normally is used in the field. For both, significant symptom remission and a reduced bacterial growth rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment increased to around eight months. This is the first report of the anti-bacterial effect of NAC against a phytopathogenic bacterium. The results obtained in this work together with the characteristics of this molecule indicate that the use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogenic bacteria. PMID:24009716

Recent evolutionary radiation and host plant specialization in the Xylella fastidiosa subspecies native to the United States.

PubMed

Nunney, Leonard; Vickerman, Danel B; Bromley, Robin E; Russell, Stephanie A; Hartman, John R; Morano, Lisa D; Stouthamer, Richard

2013-04-01

The bacterial pathogen, Xylella fastidiosa, infects many plant species in the Americas, making it a good model for investigating the genetics of host adaptation. We used multilocus sequence typing (MLST) to identify isolates of the native U.S. subsp. multiplex that were largely unaffected by intersubspecific homologous recombination (IHR) and to investigate how their evolutionary history influences plant host specialization. We identified 110 "non-IHR" isolates, 2 minimally recombinant "intermediate" ones (including the subspecific type), and 31 with extensive IHR. The non-IHR and intermediate isolates defined 23 sequence types (STs) which we used to identify 22 plant hosts (73% trees) characteristic of the subspecies. Except for almond, subsp. multiplex showed no host overlap with the introduced subspecies (subspecies fastidiosa and sandyi). MLST sequences revealed that subsp. multiplex underwent recent radiation (<25% of subspecies age) which included only limited intrasubspecific recombination (ρ/θ = 0.02); only one isolated lineage (ST50 from ash) was older. A total of 20 of the STs grouped into three loose phylogenetic clusters distinguished by nonoverlapping hosts (excepting purple leaf plum): "almond," "peach," and "oak" types. These host differences were not geographical, since all three types also occurred in California. ST designation was a good indicator of host specialization. ST09, widespread in the southeastern United States, only infected oak species, and all peach isolates were ST10 (from California, Florida, and Georgia). Only ST23 had a broad host range. Hosts of related genotypes were sometimes related, but often host groupings crossed plant family or even order, suggesting that phylogenetically plastic features of hosts affect bacterial pathogenicity.

Xylella fastidiosa infection and ethylene exposure result in xylem and water movement disruption in grapevine shoots.

PubMed

Pérez-Donoso, Alonso G; Greve, L Carl; Walton, Jeffrey H; Shackel, Ken A; Labavitch, John M

2007-02-01

It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidiosa (Xf) within xylem vessels is the sole factor responsible for the blockage of water movement in grapevines (Vitis vinifera) affected by Pierce's disease. However, results from our studies have provided substantial support for the idea that vessel obstructions, and likely other aspects of the Pierce's disease syndrome, result from the grapevine's active responses to the presence of Xf, rather than to the direct action of the bacterium. The use of magnetic resonance imaging (MRI) to observe the distribution of water within the xylem has allowed us to follow nondestructively the development of vascular system obstructions subsequent to inoculation of grapevines with Xf. Because we have hypothesized a role for ethylene produced in vines following infection, the impact of vine ethylene exposure on obstruction development was also followed using MRI. In both infected and ethylene-exposed plants, MRI shows that an important proportion of the xylem vessels become progressively air embolized after the treatments. The loss of xylem water-transporting function, assessed by MRI, has been also correlated with a decrease in stem-specific hydraulic conductivity (K(S)) and the presence of tyloses in the lumens of obstructed water conduits. We have observed that the ethylene production of leaves from infected grapevines is greater than that from healthy vines and, therefore, propose that ethylene may be involved in a series of cellular events that coordinates the vine's response to the pathogen.

The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2017-04-01

Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

Recent Evolutionary Radiation and Host Plant Specialization in the Xylella fastidiosa Subspecies Native to the United States

PubMed Central

Vickerman, Danel B.; Bromley, Robin E.; Russell, Stephanie A.; Hartman, John R.; Morano, Lisa D.; Stouthamer, Richard

2013-01-01

The bacterial pathogen, Xylella fastidiosa, infects many plant species in the Americas, making it a good model for investigating the genetics of host adaptation. We used multilocus sequence typing (MLST) to identify isolates of the native U.S. subsp. multiplex that were largely unaffected by intersubspecific homologous recombination (IHR) and to investigate how their evolutionary history influences plant host specialization. We identified 110 “non-IHR” isolates, 2 minimally recombinant “intermediate” ones (including the subspecific type), and 31 with extensive IHR. The non-IHR and intermediate isolates defined 23 sequence types (STs) which we used to identify 22 plant hosts (73% trees) characteristic of the subspecies. Except for almond, subsp. multiplex showed no host overlap with the introduced subspecies (subspecies fastidiosa and sandyi). MLST sequences revealed that subsp. multiplex underwent recent radiation (<25% of subspecies age) which included only limited intrasubspecific recombination (ρ/θ = 0.02); only one isolated lineage (ST50 from ash) was older. A total of 20 of the STs grouped into three loose phylogenetic clusters distinguished by nonoverlapping hosts (excepting purple leaf plum): “almond,” “peach,” and “oak” types. These host differences were not geographical, since all three types also occurred in California. ST designation was a good indicator of host specialization. ST09, widespread in the southeastern United States, only infected oak species, and all peach isolates were ST10 (from California, Florida, and Georgia). Only ST23 had a broad host range. Hosts of related genotypes were sometimes related, but often host groupings crossed plant family or even order, suggesting that phylogenetically plastic features of hosts affect bacterial pathogenicity. PMID:23354698

Xylella taiwanensis sp. nov., causing pear leaf scorch disease.

PubMed

Su, C-C; Deng, W-L; Jan, F-J; Chang, C-J; Huang, H; Shih, H-T; Chen, J

2016-11-01

A Gram-stain-negative, nutritionally fastidious bacterium (PLS229T) causing pear leaf scorch was identified in Taiwan and previously grouped into Xylella fastidiosa. Yet, significant variations between PLS229T and Xylellafastidiosa were noted. In this study, PLS229T was evaluated phenotypically and genotypically against representative strains of Xylellafastidiosa, including strains of the currently known subspecies of Xylellafastidiosa, Xylella fastidiosa subsp. multiplex and 'Xylella fastidiosasubsp.pauca'. Because of the difficulty of in vitro culture characterization, emphases were made to utilize the available whole-genome sequence information. The average nucleotide identity (ANI) values, an alternative for DNA-DNA hybridization relatedness, between PLS229T and Xylellafastidiosa were 83.4-83.9 %, significantly lower than the bacterial species threshold of 95 %. In contrast, sequence similarity of 16S rRNA genes was greater than 98 %, higher than the 97 % threshold to justify if two bacterial strains belong to different species. The uniqueness of PLS229T was also evident by observing only about 87 % similarity in the sequence of the 16S-23S internal transcribed spacer (ITS) between PLS229T and strains of Xylellafastidiosa, discovering significant single nucleotide polymorphisms at 18 randomly selected housekeeping gene loci, observing a distinct fatty acid profile for PLS229T compared with Xylellafastidiosa, and PLS229T having different observable phenotypes, such as different susceptibility to antibiotics. A phylogenetic tree derived from 16S rRNA gene sequences showed a distinct PLS229T phyletic lineage positioning it between Xylellafastidiosa and members of the genus Xanthomonas. On the basis of these data, a novel species, Xylella taiwanensis sp. nov. is proposed. The type strain is PLS229T (=BCRC 80915T=JCM 31187T).

Analysis of the genome-wide variations among multiple strains of the plant pathogenic bacterium Xylella fastidiosa

PubMed Central

Doddapaneni, Harshavardhan; Yao, Jiqiang; Lin, Hong; Walker, M Andrew; Civerolo, Edwin L

2006-01-01

Background The Gram-negative, xylem-limited phytopathogenic bacterium Xylella fastidiosa is responsible for causing economically important diseases in grapevine, citrus and many other plant species. Despite its economic impact, relatively little is known about the genomic variations among strains isolated from different hosts and their influence on the population genetics of this pathogen. With the availability of genome sequence information for four strains, it is now possible to perform genome-wide analyses to identify and categorize such DNA variations and to understand their influence on strain functional divergence. Results There are 1,579 genes and 194 non-coding homologous sequences present in the genomes of all four strains, representing a 76. 2% conservation of the sequenced genome. About 60% of the X. fastidiosa unique sequences exist as tandem gene clusters of 6 or more genes. Multiple alignments identified 12,754 SNPs and 14,449 INDELs in the 1528 common genes and 20,779 SNPs and 10,075 INDELs in the 194 non-coding sequences. The average SNP frequency was 1.08 × 10-2 per base pair of DNA and the average INDEL frequency was 2.06 × 10-2 per base pair of DNA. On an average, 60.33% of the SNPs were synonymous type while 39.67% were non-synonymous type. The mutation frequency, primarily in the form of external INDELs was the main type of sequence variation. The relative similarity between the strains was discussed according to the INDEL and SNP differences. The number of genes unique to each strain were 60 (9a5c), 54 (Dixon), 83 (Ann1) and 9 (Temecula-1). A sub-set of the strain specific genes showed significant differences in terms of their codon usage and GC composition from the native genes suggesting their xenologous origin. Tandem repeat analysis of the genomic sequences of the four strains identified associations of repeat sequences with hypothetical and phage related functions. Conclusion INDELs and strain specific genes have been identified as the

Sequence/structural analysis of xylem proteome emphasizes pathogenesis-related proteins, chitinases and β-1, 3-glucanases as key players in grapevine defense against Xylella fastidiosa.

PubMed

Chakraborty, Sandeep; Nascimento, Rafael; Zaini, Paulo A; Gouran, Hossein; Rao, Basuthkar J; Goulart, Luiz R; Dandekar, Abhaya M

2016-01-01

Background. Xylella fastidiosa, the causative agent of various plant diseases including Pierce's disease in the US, and Citrus Variegated Chlorosis in Brazil, remains a continual source of concern and economic losses, especially since almost all commercial varieties are sensitive to this Gammaproteobacteria. Differential expression of proteins in infected tissue is an established methodology to identify key elements involved in plant defense pathways. Methods. In the current work, we developed a methodology named CHURNER that emphasizes relevant protein functions from proteomic data, based on identification of proteins with similar structures that do not necessarily have sequence homology. Such clustering emphasizes protein functions which have multiple copies that are up/down-regulated, and highlights similar proteins which are differentially regulated. As a working example we present proteomic data enumerating differentially expressed proteins in xylem sap from grapevines that were infected with X. fastidiosa. Results. Analysis of this data by CHURNER highlighted pathogenesis related PR-1 proteins, reinforcing this as the foremost protein function in xylem sap involved in the grapevine defense response to X. fastidiosa. β-1, 3-glucanase, which has both anti-microbial and anti-fungal activities, is also up-regulated. Simultaneously, chitinases are found to be both up and down-regulated by CHURNER, and thus the net gain of this protein function loses its significance in the defense response. Discussion. We demonstrate how structural data can be incorporated in the pipeline of proteomic data analysis prior to making inferences on the importance of individual proteins to plant defense mechanisms. We expect CHURNER to be applicable to any proteomic data set.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

Phenotype overlap in Xylella fastidiosa is controlled by the cyclic di-GMP phosphodiesterase Eal in response to antibiotic exposure and diffusible signal factor-mediated cell-cell signaling.

PubMed

de Souza, Alessandra A; Ionescu, Michael; Baccari, Clelia; da Silva, Aline M; Lindow, Steven E

2013-06-01

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

Phenotype Overlap in Xylella fastidiosa Is Controlled by the Cyclic Di-GMP Phosphodiesterase Eal in Response to Antibiotic Exposure and Diffusible Signal Factor-Mediated Cell-Cell Signaling

PubMed Central

de Souza, Alessandra A.; Ionescu, Michael; Baccari, Clelia; da Silva, Aline M.

2013-01-01

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3′-5′)-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates. PMID:23542613

Comparative genomic characterization of citrus-associated Xylella fastidiosa strains.

PubMed

da Silva, Vivian S; Shida, Cláudio S; Rodrigues, Fabiana B; Ribeiro, Diógenes C D; de Souza, Alessandra A; Coletta-Filho, Helvécio D; Machado, Marcos A; Nunes, Luiz R; de Oliveira, Regina Costa

2007-12-21

The xylem-inhabiting bacterium Xylella fastidiosa (Xf) is the causal agent of Pierce's disease (PD) in vineyards and citrus variegated chlorosis (CVC) in orange trees. Both of these economically-devastating diseases are caused by distinct strains of this complex group of microorganisms, which has motivated researchers to conduct extensive genomic sequencing projects with Xf strains. This sequence information, along with other molecular tools, have been used to estimate the evolutionary history of the group and provide clues to understand the capacity of Xf to infect different hosts, causing a variety of symptoms. Nonetheless, although significant amounts of information have been generated from Xf strains, a large proportion of these efforts has concentrated on the study of North American strains, limiting our understanding about the genomic composition of South American strains - which is particularly important for CVC-associated strains. This paper describes the first genome-wide comparison among South American Xf strains, involving 6 distinct citrus-associated bacteria. Comparative analyses performed through a microarray-based approach allowed identification and characterization of large mobile genetic elements that seem to be exclusive to South American strains. Moreover, a large-scale sequencing effort, based on Suppressive Subtraction Hybridization (SSH), identified 290 new ORFs, distributed in 135 Groups of Orthologous Elements, throughout the genomes of these bacteria. Results from microarray-based comparisons provide further evidence concerning activity of horizontally transferred elements, reinforcing their importance as major mediators in the evolution of Xf. Moreover, the microarray-based genomic profiles showed similarity between Xf strains 9a5c and Fb7, which is unexpected, given the geographical and chronological differences associated with the isolation of these microorganisms. The newly identified ORFs, obtained by SSH, represent an approximately 10

Diffusible signal factor-repressed extracellular traits enable attachment of Xylella fastidiosa to insect vectors and transmission.

PubMed

Baccari, Clelia; Killiny, Nabil; Ionescu, Michael; Almeida, Rodrigo P P; Lindow, Steven E

2014-01-01

The hypothesis that a wild-type strain of Xylella fastidiosa would restore the ability of rpfF mutants blocked in diffusible signal factor production to be transmitted to new grape plants by the sharpshooter vector Graphocephala atropunctata was tested. While the rpfF mutant was very poorly transmitted by vectors irrespective of whether they had also fed on plants infected with the wild-type strain, wild-type strains were not efficiently transmitted if vectors had fed on plants infected with the rpfF mutant. About 100-fewer cells of a wild-type strain attached to wings of a vector when suspended in xylem sap from plants infected with an rpfF mutant than in sap from uninfected grapes. The frequency of transmission of cells suspended in sap from plants that were infected by the rpfF mutant was also reduced over threefold. Wild-type cells suspended in a culture supernatant of an rpfF mutant also exhibited 10-fold less adherence to wings than when suspended in uninoculated culture media. A factor released into the xylem by rpfF mutants, and to a lesser extent by the wild-type strain, thus inhibits their attachment to, and thus transmission by, sharpshooter vectors and may also enable them to move more readily through host plants.

Chitin Utilization by the Insect-Transmitted Bacterium Xylella fastidiosa▿ †

PubMed Central

Killiny, Nabil; Prado, Simone S.; Almeida, Rodrigo P. P.

2010-01-01

Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa. PMID:20656858

[Population fluctuation of sharpshooters vectors of Xylella fastidiosa Wells et al. in commercial citrus groves in northwestern Paraná State].

PubMed

Nunes, William M C; Molina, Rúbia de O; de Albuquerque, Fernando A; Corazza-Nunes, Maria J; Zanutto, Carlos A; Machado, Marcos A

2007-01-01

The citrus variegated chlorosis (CVC), an important disease of citrus in Brazil, is caused by the bacterium Xylella fastidiosa Wells et al. and transmitted by xylem-feeding sharpshooters (Hemiptera: Cicadellidae). This study evaluated the fluctuation of populations of species of sharpshooters belonging to the tribes Cicadellini and Proconiini, from subfamily Cicadelinae, in a commercial sweet orange [Citrus sinensis (L.) Osb.] grove, located in the Northwest Region of Paraná State, Brazil, in four varieties: Valência, Natal, Pêra, and Folha Murcha. Sharpshooters population was monitored using yellow stick traps sampled at 15 day-intervals, in 24 traps, from November of 1999 to March of 2004. The most abundant species were Dilobopterus costalimai Young (tribe Cicadellini) and Acrogonia citrina Marucci & Cavichioli (tribe Proconiini). Both species were detected during the complete period studied, which is important because they have great potential for transmitting CVC. Thus, since more than a sharpshooter species were detected, more efforts are recommended to monitor and control these insects in citrus groves, aiming to reduce the dissemination of CVC.

Molecular Profiling of Pierce's Disease Outlines the Response Circuitry of Vitis vinifera to Xylella fastidiosa Infection.

PubMed

Zaini, Paulo A; Nascimento, Rafael; Gouran, Hossein; Cantu, Dario; Chakraborty, Sandeep; Phu, My; Goulart, Luiz R; Dandekar, Abhaya M

2018-01-01

Pierce's disease is a major threat to grapevines caused by the bacterium Xylella fastidiosa . Although devoid of a type 3 secretion system commonly employed by bacterial pathogens to deliver effectors inside host cells, this pathogen is able to influence host parenchymal cells from the xylem lumen by secreting a battery of hydrolytic enzymes. Defining the cellular and biochemical changes induced during disease can foster the development of novel therapeutic strategies aimed at reducing the pathogen fitness and increasing plant health. To this end, we investigated the transcriptional, proteomic, and metabolomic responses of diseased Vitis vinifera compared to healthy plants. We found that several antioxidant strategies were induced, including the accumulation of gamma-aminobutyric acid (GABA) and polyamine metabolism, as well as iron and copper chelation, but these were insufficient to protect the plant from chronic oxidative stress and disease symptom development. Notable upregulation of phytoalexins, pathogenesis-related proteins, and various aromatic acid metabolites was part of the host responses observed. Moreover, upregulation of various cell wall modification enzymes followed the proliferation of the pathogen within xylem vessels, consistent with the intensive thickening of vessels' secondary walls observed by magnetic resonance imaging. By interpreting the molecular profile changes taking place in symptomatic tissues, we report a set of molecular markers that can be further explored to aid in disease detection, breeding for resistance, and developing therapeutics.

Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

PubMed

Machado, Agnes Thiane Pereira; Fonseca, Emanuella Maria Barreto; Reis, Marcelo Augusto Dos; Saraiva, Antonio Marcos; Santos, Clelton Aparecido Dos; de Toledo, Marcelo Augusto Szymanski; Polikarpov, Igor; de Souza, Anete Pereira; Aparicio, Ricardo; Iulek, Jorge

2017-10-01

Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein. © 2017 Wiley Periodicals, Inc.

Differences in Stylet Penetration Behaviors of Glassy-winged Sharpshooters on Xylella-Resistant Vitis candicans vs. Susceptible Vitis vinifera cv. ‘Chardonnay’

USDA-ARS?s Scientific Manuscript database

Electrical penetration graph (EPG) monitoring was used to compare stylet penetration behaviors of glassy-winged sharpshooter (GWSS), a vector of Xylella fastidiosa (Xf), on Xf-resistant Vitis candicans grape vs. susceptible V. vinifera cv. ‘Chardonnay.’ Frequency of occurrence of X waves (represent...

Molecular Profiling of Pierce’s Disease Outlines the Response Circuitry of Vitis vinifera to Xylella fastidiosa Infection

PubMed Central

Zaini, Paulo A.; Nascimento, Rafael; Gouran, Hossein; Cantu, Dario; Chakraborty, Sandeep; Phu, My; Goulart, Luiz R.; Dandekar, Abhaya M.

2018-01-01

Pierce’s disease is a major threat to grapevines caused by the bacterium Xylella fastidiosa. Although devoid of a type 3 secretion system commonly employed by bacterial pathogens to deliver effectors inside host cells, this pathogen is able to influence host parenchymal cells from the xylem lumen by secreting a battery of hydrolytic enzymes. Defining the cellular and biochemical changes induced during disease can foster the development of novel therapeutic strategies aimed at reducing the pathogen fitness and increasing plant health. To this end, we investigated the transcriptional, proteomic, and metabolomic responses of diseased Vitis vinifera compared to healthy plants. We found that several antioxidant strategies were induced, including the accumulation of gamma-aminobutyric acid (GABA) and polyamine metabolism, as well as iron and copper chelation, but these were insufficient to protect the plant from chronic oxidative stress and disease symptom development. Notable upregulation of phytoalexins, pathogenesis-related proteins, and various aromatic acid metabolites was part of the host responses observed. Moreover, upregulation of various cell wall modification enzymes followed the proliferation of the pathogen within xylem vessels, consistent with the intensive thickening of vessels’ secondary walls observed by magnetic resonance imaging. By interpreting the molecular profile changes taking place in symptomatic tissues, we report a set of molecular markers that can be further explored to aid in disease detection, breeding for resistance, and developing therapeutics.

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

PubMed Central

Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W.; Almeida-Souza, Hebréia O.; Tu, Aye; Rao, Basuthkar J.; Feldstein, Paul A.; Bruening, George; Goulart, Luiz R.; Dandekar, Abhaya M.

2016-01-01

Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms. PMID:26753904

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines.

PubMed

Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W; Almeida-Souza, Hebréia O; Tu, Aye; Rao, Basuthkar J; Feldstein, Paul A; Bruening, George; Goulart, Luiz R; Dandekar, Abhaya M

2016-01-12

Pierce's disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

Enhanced Reliability and Accuracy for Field Deployable Bioforensic Detection and Discrimination of Xylella fastidiosa subsp. pauca, Causal Agent of Citrus Variegated Chlorosis Using Razor Ex Technology and TaqMan Quantitative PCR

PubMed Central

Fletcher, Jacqueline; Melcher, Ulrich; Ochoa Corona, Francisco Manuel

2013-01-01

A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets. PMID:24312333

Enhanced reliability and accuracy for field deployable bioforensic detection and discrimination of Xylella fastidiosa subsp. pauca, causal agent of citrus variegated chlorosis using razor ex technology and TaqMan quantitative PCR.

PubMed

Ouyang, Ping; Arif, Mohammad; Fletcher, Jacqueline; Melcher, Ulrich; Ochoa Corona, Francisco Manuel

2013-01-01

A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.

Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca.

PubMed

Giampetruzzi, Annalisa; Morelli, Massimiliano; Saponari, Maria; Loconsole, Giuliana; Chiumenti, Michela; Boscia, Donato; Savino, Vito N; Martelli, Giovanni P; Saldarelli, Pasquale

2016-06-27

The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. A global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. Collectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies

Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-L-glutamate synthase/kinase with and without a His tag bound to N-acetyl-L-glutamate.

PubMed

Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

2015-01-01

Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P4(1)2(1)2, with unit-cell parameters a=b=51.72, c=242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a=63.48, b=122.34, c=75.88 Å, β=107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K.

Mu-Like Prophage in Serogroup B Neisseria meningitidis Coding for Surface-Exposed Antigens

PubMed Central

Masignani, Vega; Giuliani, Marzia Monica; Tettelin, Hervé; Comanducci, Maurizio; Rappuoli, Rino; Scarlato, Vincenzo

2001-01-01

Sequence analysis of the genome of Neisseria meningititdis serogroup B revealed the presence of an ∼35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of Escherichia coli Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in Haemophilus influenzae. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies. PMID:11254622

Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission.

PubMed

Chatterjee, Subhadeep; Killiny, Nabil; Almeida, Rodrigo P P; Lindow, Steven E

2010-10-01

Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

Fatty Acid Methyl Ester (FAME) analyses for characterization and detection of grapevine pathogens

USDA-ARS?s Scientific Manuscript database

Grapevines can become infected by a variety of devastating pathogens, including the bacterium Xylella fastidiosa and canker fungi. Multiple strains of Xylella fastidiosa exist, each causing different diseases on various hosts. Although sequence-based genotyping can assist in distinguishing these str...

The temperate marine phage PhiHAP-1 of Halomonas aquamarina possesses a linear plasmid-like prophage genome.

PubMed

Mobberley, Jennifer M; Authement, R Nathan; Segall, Anca M; Paul, John H

2008-07-01

A myovirus-like temperate phage, PhiHAP-1, was induced with mitomycin C from a Halomonas aquamarina strain isolated from surface waters in the Gulf of Mexico. The induced cultures produced significantly more virus-like particles (VLPs) (3.73 x 10(10) VLP ml(-1)) than control cultures (3.83 x 10(7) VLP ml(-1)) when observed with epifluorescence microscopy. The induced phage was sequenced by using linker-amplified shotgun libraries and contained a genome 39,245 nucleotides in length with a G+C content of 59%. The PhiHAP-1 genome contained 46 putative open reading frames (ORFs), with 76% sharing significant similarity (E value of <10(-3)) at the protein level with other sequences in GenBank. Putative functional gene assignments included small and large terminase subunits, capsid and tail genes, an N6-DNA adenine methyltransferase, and lysogeny-related genes. Although no integrase was found, the PhiHAP-1 genome contained ORFs similar to protelomerase and parA genes found in linear plasmid-like phages with telomeric ends. Southern probing and PCR analysis of host genomic, plasmid, and PhiHAP-1 DNA indicated a lack of integration of the prophage with the host chromosome and a difference in genome arrangement between the prophage and virion forms. The linear plasmid prophage form of PhiHAP-1 begins with the protelomerase gene, presumably due to the activity of the protelomerase, while the induced phage particle has a circularly permuted genome that begins with the terminase genes. The PhiHAP-1 genome shares synteny and gene similarity with coliphage N15 and vibriophages VP882 and VHML, suggesting an evolutionary heritage from an N15-like linear plasmid prophage ancestor.

A TaqMan-based real time PCR assay for specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander.

PubMed

Guan, Wei; Shao, Jonathan; Singh, Raghuwinder; Davis, Robert E; Zhao, Tingchang; Huang, Qi

2013-02-15

A TaqMan-based real-time PCR assay was developed for specific detection of strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences found only in the genome of oleander strain Ann1. The assay is specific, allowing detection of only oleander-infecting strains, not other strains of X. fastidiosa nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4fg DNA of X. fastidiosa per reaction in vitro and in planta. The assay can also be applied to detect low numbers of X. fastidiosa in insect samples, or further developed into a multiplex real-time PCR assay to simultaneously detect and distinguish diverse strains of X. fastidiosa that may occupy the same hosts or insect vectors. Specific and sensitive detection and quantification of oleander strains of X. fastidiosa should be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs. Published by Elsevier B.V.

Comparative analysis of prophages in Streptococcus mutans genomes

PubMed Central

Fu, Tiwei; Fan, Xiangyu; Long, Quanxin; Deng, Wanyan; Song, Jinlin

2017-01-01

Prophages have been considered genetic units that have an intimate association with novel phenotypic properties of bacterial hosts, such as pathogenicity and genomic variation. Little is known about the genetic information of prophages in the genome of Streptococcus mutans, a major pathogen of human dental caries. In this study, we identified 35 prophage-like elements in S. mutans genomes and performed a comparative genomic analysis. Comparative genomic and phylogenetic analyses of prophage sequences revealed that the prophages could be classified into three main large clusters: Cluster A, Cluster B, and Cluster C. The S. mutans prophages in each cluster were compared. The genomic sequences of phismuN66-1, phismuNLML9-1, and phismu24-1 all shared similarities with the previously reported S. mutans phages M102, M102AD, and ϕAPCM01. The genomes were organized into seven major gene clusters according to the putative functions of the predicted open reading frames: packaging and structural modules, integrase, host lysis modules, DNA replication/recombination modules, transcriptional regulatory modules, other protein modules, and hypothetical protein modules. Moreover, an integrase gene was only identified in phismuNLML9-1 prophages. PMID:29158986

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.

PubMed

Gouran, Hossein; Chakraborty, Sandeep; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

PubMed Central

Rao, Basuthkar J.; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction. PMID:25717364

Identification of Bacterial Plant Pathogens Using Multilocus Polymerase Chain Reaction/Electrospray Ionization-Mass Spectrometry

DTIC Science & Technology

2008-01-01

sandii FK-53; OLF#1 Oleander; USA 1 Xylophilus ampelinus FB-1178 Grape; S. Africa 1 Xylophilus ampelinus FJ-3; 60002 Grape; S. Africa 1 1160...campestris Xanthomonas campestris Xylella fastidiosa (6 strains) Xylella fastidiosa Xylophilus ampelinus (2 strains) Xylophilus ampelinus ...Rathayibacter iranicus Rathayibacter iranicus Xylophilus ampelinus Xylophilus ampelinus a Purified DNAs from multiple bacteria were mixed at equal

The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" encodes an afp-like prophage possibly used for protein secretion.

PubMed

Penz, Thomas; Horn, Matthias; Schmitz-Esser, Stephan

2010-01-01

The recently sequenced genome of the obligate intracellular amoeba symbiont 'Candidatus Amoebophilus asiaticus' is unique among prokaryotic genomes due to its extremely large fraction of genes encoding proteins harboring eukaryotic domains such as ankyrin-repeats, TPR/SEL1 repeats, leucine-rich repeats, as well as F- and U-box domains, most of which likely serve in the interaction with the amoeba host. Here we provide evidence for the presence of additional proteins which are presumably presented extracellularly and should thus also be important for host cell interaction. Surprisingly, we did not find homologues of any of the well-known protein secretion systems required to translocate effector proteins into the host cell in the A. asiaticus genome, and the type six secretion systems seems to be incomplete. Here we describe the presence of a putative prophage in the A. asiaticus genome, which shows similarity to the antifeeding prophage from the insect pathogen Serratia entomophila. In S. entomophila this system is used to deliver toxins into insect hosts. This putative antifeeding-like prophage might thus represent the missing protein secretion apparatus in A. asiaticus.

Enterococcus faecalis Prophage Dynamics and Contributions to Pathogenic Traits

PubMed Central

Matos, Renata C.; Lapaque, Nicolas; Rigottier-Gois, Lionel; Debarbieux, Laurent; Meylheuc, Thierry; Gonzalez-Zorn, Bruno; Repoila, Francis; Lopes, Maria de Fatima; Serror, Pascale

2013-01-01

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among

The role of prophage for genome diversification within a clonal lineage of Lactobacillus johnsonii: characterization of the defective prophage LJ771.

PubMed

Denou, Emmanuel; Pridmore, Raymond David; Ventura, Marco; Pittet, Anne-Cécile; Zwahlen, Marie-Camille; Berger, Bernard; Barretto, Caroline; Panoff, Jean-Michel; Brüssow, Harald

2008-09-01

Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. These isolates belonged to the same clonal lineage and differed mainly by a 40.8-kb prophage, LJ771, belonging to the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as an integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE ("antitoxin")/pemK ("toxin") gene cassette was detected in LJ771 but not in the L. gasseri prophages. Expressed pemK could be cloned in Escherichia coli only together with the mazE gene. LJ771 was shown to be highly stable and could be cured only by coexpression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complemented the 5' end of this gene, creating a protein with a slightly altered N-terminal sequence. The two L. johnsonii strains had identical in vitro growth and in vivo gut persistence phenotypes. Also, in an isogenic background, the presence of the prophage resulted in no growth disadvantage.

Grapevine rootstock effects on scion sap phenolic levels, resistance to Xylella fastidiosa infection, and progression of Pierce's disease

PubMed Central

Wallis, Christopher M.; Wallingford, Anna K.; Chen, Jianchi

2013-01-01

The xylem-limited bacterium Xylella fastidiosa (Xf) causes Pierce's disease (PD), an important disease of grapevine, Vitis vinifera L. Grapevine rootstocks were developed to provide increased resistance to root disease, but rootstock effects on cane and vine diseases remain unclear. Grapevines that consisted of Cabernet Sauvignon or Chardonnay grafted to 13 different rootstocks were inoculated with Xf and evaluated for PD severity and Xf titer after 6 months. A subset of six rootstock/scion combinations had xylem sap phenolic levels assessed in non-infected and Xf-infected grapevines. Vigor also was analyzed by measuring root lengths and masses. Cabernet Sauvignon grafted to 101-14MG, 1103P, 420A, or Schwarzmann had reduced PD severity compared to Cabernet Sauvignon grafted to 110R, 5BB, or SO4. Chardonnay grafted to Salt Creek or Freedom had reduced PD severity compared to Chardonnay grafted to RS3 or Schwarzmann. Chardonnay grafted to RS3 had greater Xf titer than Chardonnay grafted to 101-14MG, Freedom, or Salt Creek. No other differences in Xf titer among rootstocks were observed. Of the six scion/rootstock combinations which had xylem sap phenolics analyzed, Chardonnay/RS3 had the highest levels of most phenolics whereas Cabernet Sauvignon/101-14MG had the lowest phenolic levels. However, Chardonnay/101-14MG, which had mild PD symptoms, had greater sap levels of caftaric acid than other scion/rootstock combinations. Sap levels of caftaric acid, methyl salicylate, a procyanidin trimer, and quinic acid were greater in Xf-infected vs. non-infected grapevines. Chardonnay on 101-14MG or Salt Creek had greater root mass than Chardonnay on RS3. Cabernet Sauvignon on 101-14MG had greater root mass than Cabernet Sauvignon on 110R. These results identified rootstocks with the capacity for reducing PD symptom progression. Rootstocks also were shown to affect Xf titer, xylem sap phenolic levels, and plant vigor. PMID:24376452

Combined use of a new SNP-based assay and multilocus SSR markers to assess genetic diversity of Xylella fastidiosa subsp. pauca infecting citrus and coffee plants.

PubMed

Montes-Borrego, Miguel; Lopes, Joao R S; Jiménez-Díaz, Rafael M; Landa, Blanca B

2015-03-01

Two haplotypes of Xylella fastidiosa subsp. pauca (Xfp) that correlated with their host of origin were identified in a collection of 90 isolates infecting citrus and coffee plants in Brazil, based on a single-nucleotide polymorphism in the gyrB sequence. A new single-nucleotide primer extension (SNuPE) protocol was designed for rapid identification of Xfp according to the host source. The protocol proved to be robust for the prediction of the Xfp host source in blind tests using DNA from cultures of the bacterium, infected plants, and insect vectors allowed to feed on Xfp-infected citrus plants. AMOVA and STRUCTURE analyses of microsatellite data separated most Xfp populations on the basis of their host source, indicating that they were genetically distinct. The combined use of the SNaPshot protocol and three previously developed multilocus SSR markers showed that two haplotypes and distinct isolates of Xfp infect citrus and coffee in Brazil and that multiple, genetically different isolates can be present in a single orchard or infect a single tree. This combined approach will be very useful in studies of the epidemiology of Xfp-induced diseases, host specificity of bacterial genotypes, the occurrence of Xfp host jumping, vector feeding habits, etc., in economically important cultivated plants or weed host reservoirs of Xfp in Brazil and elsewhere. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Invasive Threats to the American Homeland

DTIC Science & Technology

2004-04-01

The second example is the glassy-winged sharpshooter, an invasive insect that hosts the bacterium Xylella Fastidiosa . The insect was first de- tected... Xylella Fastidiosa causes Pierce’s Disease in grapes, which infects and kills the grapevine. The glassy-winged sharpshooter transmits and spreads the...netic processes.” Adversaries of the United States may modify the genetics of an invasive species to increase its competitiveness, virulence , lethality

Invasive Species - A Threat to the Homeland?

DTIC Science & Technology

2003-04-07

sharpshooter, an invasive insect that hosts the bacterium Xylella Fastidiosa . The insect was first detected in California in 1990. Although it is...uncertain how it arrived in California, it is believed to have arrived on plants imported from an infected area. The bacterium Xylella Fastidiosa causes...United States may modify the genetics of an invasive species to increase its competitiveness, virulence , lethality, or resistance to control measures

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

PubMed

Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

Infectivity and transmission of Xylella fastidiosa Salento strain by Philaenus spumarius L. (Hemiptera: Aphrophoridae) in Apulia, Italy

USDA-ARS?s Scientific Manuscript database

Discovery of X. fastidiosa from olive trees with “Olive quick decline syndrome" (OQDS) in October 2013 on the western coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards was the meado...

A Simple Defined Medium for the Production of True Diketopiperazines in Xylella fastidiosa and Their Identification by Ultra-Fast Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

PubMed

Silva, Michelli Massaroli da; Andrade, Moacir Dos Santos; Bauermeister, Anelize; Merfa, Marcus Vinícius; Forim, Moacir Rossi; Fernandes, João Batista; Vieira, Paulo Cezar; Silva, Maria Fátima das Graças Fernandes da; Lopes, Norberto Peporine; Machado, Marcos Antônio; Souza, Alessandra Alves de

2017-06-13

Diketopiperazines can be generated by non-enzymatic cyclization of linear dipeptides at extreme temperature or pH, and the complex medium used to culture bacteria and fungi including phytone peptone and trypticase peptone, can also produce cyclic peptides by heat sterilization. As a result, it is not always clear if many diketopiperazines reported in the literature are artifacts formed by the different complex media used in microorganism growth. An ideal method for analysis of these compounds should identify whether they are either synthesized de novo from the products of primary metabolism and deliver true diketopiperazines. A simple defined medium ( X. fastidiosa medium or XFM) containing a single carbon source and no preformed amino acids has emerged as a method with a particularly high potential for the grown of X. fastidiosa and to produce genuine natural products. In this work, we identified a range of diketopiperazines from X. fastidiosa 9a5c growth in XFM, using Ultra-Fast Liquid Chromatography coupled with mass spectrometry. Diketopiperazines are reported for the first time from X. fastidiosa , which is responsible for citrus variegated chlorosis. We also report here fatty acids from X. fastidiosa , which were not biologically active as diffusible signals, and the role of diketopiperazines in signal transduction still remains unknown.

Anterior foregut microbiota of the glassy-winged sharpshooter explored using deep 16S rRNA gene sequencing from individual insects

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce’s disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium) of sharpshooters, where ...

Exploring glassy-winged sharpshooter microbiota using deep 16S rRNA sequencing from individual insects

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce’s disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium) of sharpshooters, where ...

Glassy-winged sharpshooter Microbiota explored using deep 16S rRNA sequencing from individual insects

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce’s disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium) of sharpshooters, where ...

Plant water stress effects on the net dispersal rate of the insect vector Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) and movement of its egg parasitoid, Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae)

USDA-ARS?s Scientific Manuscript database

Homalodisca vitripennis, one of the main vectors of Xylella fastidiosa, is associated with citrus plantings in California, USA. Infested citrus orchards act as a source of vectors to adjacent vineyards where X. fastidiosa causes Pierce’s disease (PD). An analysis of the pattern and rate of movement ...

Predominance of Single Prophage Carrying a CRISPR/cas System in "Candidatus Liberibacter asiaticus" Strains in Southern China.

PubMed

Zheng, Zheng; Bao, Minli; Wu, Fengnian; Chen, Jianchi; Deng, Xiaoling

2016-01-01

"Candidatus Liberibacter asiaticus" (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the "Ca. Liberibacter" genera.

Rootstock effects on almond leaf scorch disease incidence and severity

USDA-ARS?s Scientific Manuscript database

A five-year field study was conducted to evaluate effects of duration and exclusion of Xylella fastidiosa infections on young almond tree performance and their links to tree vigor. ‘Nemaguard’, ‘Okinawa’, ‘Nonpareil’, and Y119 were used as rootstocks for almond scion ‘Sonora’. Among X.fastidiosa-inf...

Predominance of Single Prophage Carrying a CRISPR/cas System in “Candidatus Liberibacter asiaticus” Strains in Southern China

PubMed Central

Zheng, Zheng; Bao, Minli; Wu, Fengnian; Chen, Jianchi; Deng, Xiaoling

2016-01-01

“Candidatus Liberibacter asiaticus” (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the “Ca. Liberibacter” genera. PMID:26741827

Association of the sharpshooter X wave with xylem inoculation of Xylella fastidiosa leading to systemic, symptomatic Pierce’s diesease infection in grape

USDA-ARS?s Scientific Manuscript database

Despite several decades of study, the mechanism of inoculation of X. fastidiosa (Xf) to grapevines by its sharpshooter vectors still is not fully understood. Recent research showed that Xf is inoculated into or onto artificial diets by a combination of egestion and salivation. However, the salivatio...

Temperate Phages Acquire DNA from Defective Prophages by Relaxed Homologous Recombination: The Role of Rad52-Like Recombinases

PubMed Central

De Paepe, Marianne; Hutinet, Geoffrey; Son, Olivier; Amarir-Bouhram, Jihane; Schbath, Sophie; Petit, Marie-Agnès

2014-01-01

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage λ, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like

Assessing the functionality and genetic diversity of lactococcal prophages.

PubMed

Kelleher, Philip; Mahony, Jennifer; Schweinlin, Katharina; Neve, Horst; Franz, Charles M; van Sinderen, Douwe

2018-05-02

Lactococcus lactis is a lactic acid bacterium that is intensively and globally exploited in commercial dairy food fermentations. Though the presence of prophages in lactococcal genomes is widely reported, only limited studies pertaining to the stability of prophages in lactococcal genomes have been performed. The current study reports on the complete genome exploration of thirty lactococcal strains for the presence of potentially intact prophages, so as to assess their genomic diversity and the associated risk or benefit of harbouring such prophages. Genomic predictions partnered with mitomycin C inductions and flow cytometric analysis of the induced cell lysates confirmed that only four strains consistently produced intact phage particles, thus indicating a relatively low risk associated with prophage induction in the fermentation setting. Our analysis revealed the widespread presence of putative phage-resistance systems encoded by lactococcal prophages, thus highlighting the potential benefits for host fitness. Many of the identified lactococcal prophages belong to the so-called P335 phage group, while a large group of phage remnants bear similarity to members of the 936 phage group. The P335 phage group was recently shown to encompass four distinct genetic lineages. Our study identified an additional lineage, thus expanding the diversity of this industrially significant phage group. Copyright © 2018 Elsevier B.V. All rights reserved.

Evolutionary Genomics of an Ancient Prophage of the Order Sphingomonadales

PubMed Central

Viswanathan, Vandana; Narjala, Anushree; Ravichandran, Aravind; Jayaprasad, Suvratha

2017-01-01

The order Sphingomonadales, containing the families Erythrobacteraceae and Sphingomonadaceae, is a relatively less well-studied phylogenetic branch within the class Alphaproteobacteria. Prophage elements are present in most bacterial genomes and are important determinants of adaptive evolution. An “intact” prophage was predicted within the genome of Sphingomonas hengshuiensis strain WHSC-8 and was designated Prophage IWHSC-8. Loci homologous to the region containing the first 22 open reading frames (ORFs) of Prophage IWHSC-8 were discovered among the genomes of numerous Sphingomonadales. In 17 genomes, the homologous loci were co-located with an ORF encoding a putative superoxide dismutase. Several other lines of molecular evidence implied that these homologous loci represent an ancient temperate bacteriophage integration, and this horizontal transfer event pre-dated niche-based speciation within the order Sphingomonadales. The “stabilization” of prophages in the genomes of their hosts is an indicator of “fitness” conferred by these elements and natural selection. Among the various ORFs predicted within the conserved prophages, an ORF encoding a putative proline-rich outer membrane protein A was consistently present among the genomes of many Sphingomonadales. Furthermore, the conserved prophages in six Sphingomonas sp. contained an ORF encoding a putative spermidine synthase. It is possible that one or more of these ORFs bestow selective fitness, and thus the prophages continue to be vertically transferred within the host strains. Although conserved prophages have been identified previously among closely related genera and species, this is the first systematic and detailed description of orthologous prophages at the level of an order that contains two diverse families and many pigmented species. PMID:28201618

Comparative Genomics and Transcriptional Analysis of Prophages Identified in the Genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei†

PubMed Central

Ventura, Marco; Canchaya, Carlos; Bernini, Valentina; Altermann, Eric; Barrangou, Rodolphe; McGrath, Stephen; Claesson, Marcus J.; Li, Yin; Leahy, Sinead; Walker, Carey D.; Zink, Ralf; Neviani, Erasmo; Steele, Jim; Broadbent, Jeff; Klaenhammer, Todd R.; Fitzgerald, Gerald F.; O'Toole, Paul W.; van Sinderen, Douwe

2006-01-01

Lactobacillus gasseri ATCC 33323, Lactobacillus salivarius subsp. salivarius UCC 118, and Lactobacillus casei ATCC 334 contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1) distinguishable prophage sequences, respectively. Sequence analysis revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group of Sfi11-like pac site and cos site Siphoviridae, respectively. Phylogenetic investigation of these newly described prophage sequences revealed that they have not followed an evolutionary development similar to that of their bacterial hosts and that they show a high degree of diversity, even within a species. The attachment sites were determined for all these prophage elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage Sal2 integrates in a predicted arginino-succinate lyase-encoding gene. In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are integrated in noncoding regions in the L. casei ATCC 334 and L. salivarius UCC 118 genomes. Northern analysis showed that large parts of the prophage genomes are transcriptionally silent and that transcription is limited to genome segments located near the attachment site. Finally, pulsed-field gel electrophoresis followed by Southern blot hybridization with specific prophage probes indicates that these prophage sequences are narrowly distributed within lactobacilli. PMID:16672450

Quantification and localization of hesperidin and rutin in Citrus sinensis grafted on C. limonia after Xylella fastidiosa infection by HPLC-UV and MALDI imaging mass spectrometry.

PubMed

Soares, Márcio Santos; da Silva, Danielle Fernandes; Forim, Moacir Rossi; da Silva, Maria Fátima das Graças Fernandes; Fernandes, João Batista; Vieira, Paulo Cezar; Silva, Denise Brentan; Lopes, Norberto Peporine; de Carvalho, Sérgio Alves; de Souza, Alessandra Alves; Machado, Marcos Antônio

2015-07-01

A high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed for quantifying hesperidin and rutin levels in leaves and stems of Citrus limonia, with a good linearity over a range of 1.0-80.0 and 1.0-50.0 μg mL(-1) respectively, with r(2)>0.999 for all curves. The limits of detection (LOD) for both flavonoids were 0.6 and 0.5 μg mL(-1), respectively, with quantification (LOQ) being 2.0 and 1.0 μg mL(-1), respectively. The quantification method was applied to Citrus sinensis grafted onto C. limonia with and without CVC (citrus variegated chlorosis) symptoms after Xylella fastidiosa infection. The total content of rutin was low and practically constant in all analyses in comparison with hesperidin, which showed a significant increase in its amount in symptomatic leaves. Scanning electron microscopy studies on leaves with CVC symptoms showed vessel occlusion by biofilm, and a crystallized material was noted. Considering the difficulty in isolating these crystals for analysis, tissue sections were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to confirm the presence of hesperidin at the site of infection. The images constructed from MS/MS data with a specific diagnostic fragment ion (m/z 483) also showed higher ion intensities for it in infected plants than in healthy ones, mainly in the vessel regions. These data suggest that hesperidin plays a role in the plant-pathogen interaction, probably as a phytoanticipin. This method was also applied to C. sinensis and C. limonia seedlings, and comparison with the graft results showed that the rootstock had an increased hesperidin content ∼3.6 fold greater in the graft stem than in the stem of C. sinensis seedlings. Increase in hesperidin content by rootstock can be related to induced internal defense mechanisms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prophinder: a computational tool for prophage prediction in prokaryotic genomes.

PubMed

Lima-Mendez, Gipsi; Van Helden, Jacques; Toussaint, Ariane; Leplae, Raphaël

2008-03-15

Prophinder is a prophage prediction tool coupled with a prediction database, a web server and web service. Predicted prophages will help to fill the gaps in the current sparse phage sequence space, which should cover an estimated 100 million species. Systematic and reliable predictions will enable further studies of prophages contribution to the bacteriophage gene pool and to better understand gene shuffling between prophages and phages infecting the same host. Softare is available at http://aclame.ulb.ac.be/prophinder

Filamentation and spatiotemporal distribution of extracellular polymeric substances: role on X.fastidiosa single cell adhesion and biofilm formation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Janissen, Richard; Murillo, Duber M.; Niza, Barbara; Sahoo, Prasana K.; Monteiro, Moniellen P.; César, Carlos L.; Carvalho, Hernandes F.; de Souza, Alessandra A.; Cotta, Monica A.

2016-04-01

Biofilms can be defined as a community of microorganisms attached to a surface, living embedded in a self- produced matrix of hydrated extracellular polymeric substances (EPS) which comprises most of the biofilm mass. We have recently used an extensive pool of microscopy techniques (confocal fluorescence, electron and scanning probe microscopies) at the micro and nanoscales in order to create a detailed temporal observation of Xylella fastidiosa biofilm formation, using both wild type strain and Green Fluorescent Protein (GFP)-modified cells of this citrus phytopathogen. We have identified three different EPS compositions, as well as their spatial and temporal distribution from single cell to mature biofilm formation stages. In the initial adhesion stage, soluble-EPS (S-EPS) accumulates at cell polar regions and forms a surface layer which facilitates irreversible cell attachment and cell cluster formation. These small clusters are subsequently connected by filamentous cells; further S-EPS surface coverage facilitates cell attachment and form filaments, leading to a floating framework of mature biofilms. The important role of EPS in X.fastidiosa biology was further investigated by imunolabelling experiments to detect the distribution of XadA1 adhesin, which is expressed in early stages of biofilm formation and released in outer membrane vesicles. This protein is located mainly in S-EPS covered areas, as well as on the filaments, indicating a molecular pathway to the enhanced cell attachment previously observed. These results suggest that S-EPS may thus represent an important target for disease control, slow plant colonization by the bacteria, keeping the plant more productive in the field.

A New (Type 3) Prophage of “Candidatus Liberibacter asiaticus” in China

USDA-ARS?s Scientific Manuscript database

Prophages are important genetic entities of “Candidatus Liberibacter asiaticus” (CLas), a non-culturable a-proteobacterium associated with citrus Huanglongbing (HLB). Two CLas prophages have been described, SC1 (NC_019549.1, Type 1) and SC2 (NC_019550.1, Type 2). To explore the prophage repertoire, ...

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food.

PubMed

Fang, Yuan; Mercer, Ryan G; McMullen, Lynn M; Gänzle, Michael G

2017-10-01

The prophage-encoded Shiga toxin is a major virulence factor in Stx-producing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, food-related stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 Δ stx2 :: gfp :: amp r In addition, the effect of stress on production of phage particles was determined. The lethality of stressors, including heat, HCl, lactic acid, hydrogen peroxide, and high hydrostatic pressure, was selected to reduce cell counts by 1 to 2 log CFU/ml. The integrity of the bacterial membrane after exposure to stress was measured by propidium iodide (PI). The fluorescent signals of green fluorescent protein (GFP) and PI were quantified by flow cytometry. The mechanism of prophage induction by stress was evaluated by relative gene expression of recA and cell morphology. Acid (pH < 3.5) and H 2 O 2 (2.5 mM) induced the expression of stx 2 in about 18% and 3% of the population, respectively. The mechanism of prophage induction by acid differs from that of induction by H 2 O 2 H 2 O 2 induction but not acid induction corresponded to production of infectious phage particles, upregulation of recA , and cell filamentation. Pressure (200 MPa) or heat did not induce the Stx2-encoding prophage (Stx2-prophage). Overall, the quantification method developed in this study allowed investigation of prophage induction and physiological properties at the single-cell level. H 2 O 2 and acids mediate different pathways to induce Stx2-prophage. IMPORTANCE Induction of the Stx-prophage in STEC results in production of phage particles and Stx and thus relates to virulence as well as the transduction of virulence genes. This study developed a method for a detection of the induction of Stx-prophages at the

Induction of Shiga Toxin-Encoding Prophage by Abiotic Environmental Stress in Food

PubMed Central

Fang, Yuan; Mercer, Ryan G.; McMullen, Lynn M.

2017-01-01

ABSTRACT The prophage-encoded Shiga toxin is a major virulence factor in Stx-producing Escherichia coli (STEC). Toxin production and phage production are linked and occur after induction of the RecA-dependent SOS response. However, food-related stress and Stx-prophage induction have not been studied at the single-cell level. This study investigated the effects of abiotic environmental stress on stx expression by single-cell quantification of gene expression in STEC O104:H4 Δstx2::gfp::ampr. In addition, the effect of stress on production of phage particles was determined. The lethality of stressors, including heat, HCl, lactic acid, hydrogen peroxide, and high hydrostatic pressure, was selected to reduce cell counts by 1 to 2 log CFU/ml. The integrity of the bacterial membrane after exposure to stress was measured by propidium iodide (PI). The fluorescent signals of green fluorescent protein (GFP) and PI were quantified by flow cytometry. The mechanism of prophage induction by stress was evaluated by relative gene expression of recA and cell morphology. Acid (pH < 3.5) and H2O2 (2.5 mM) induced the expression of stx2 in about 18% and 3% of the population, respectively. The mechanism of prophage induction by acid differs from that of induction by H2O2. H2O2 induction but not acid induction corresponded to production of infectious phage particles, upregulation of recA, and cell filamentation. Pressure (200 MPa) or heat did not induce the Stx2-encoding prophage (Stx2-prophage). Overall, the quantification method developed in this study allowed investigation of prophage induction and physiological properties at the single-cell level. H2O2 and acids mediate different pathways to induce Stx2-prophage. IMPORTANCE Induction of the Stx-prophage in STEC results in production of phage particles and Stx and thus relates to virulence as well as the transduction of virulence genes. This study developed a method for a detection of the induction of Stx-prophages at the single

A prophage tail-like protein is deployed by Burkholderia bacteria to feed on fungi.

PubMed

Swain, Durga Madhab; Yadav, Sunil Kumar; Tyagi, Isha; Kumar, Rahul; Kumar, Rajeev; Ghosh, Srayan; Das, Joyati; Jha, Gopaljee

2017-09-01

Some bacteria can feed on fungi, a phenomenon known as mycophagy. Here we show that a prophage tail-like protein (Bg_9562) is essential for mycophagy in Burkholderia gladioli strain NGJ1. The purified protein causes hyphal disintegration and inhibits growth of several fungal species. Disruption of the Bg_9562 gene abolishes mycophagy. Bg_9562 is a potential effector secreted by a type III secretion system (T3SS) and is translocated into fungal mycelia during confrontation. Heterologous expression of Bg_9562 in another bacterial species, Ralstonia solanacearum, confers mycophagous ability in a T3SS-dependent manner. We propose that the ability to feed on fungi conferred by Bg_9562 may help the bacteria to survive in certain ecological niches. Furthermore, considering its broad-spectrum antifungal activity, the protein may be potentially useful in biotechnological applications to control fungal diseases.Some bacteria can feed on live fungi through unclear mechanisms. Here, the authors show that a T3SS-secreted protein, which is homologous to phage tail proteins, allows a Burkholderia gladioli strain to kill and feed on various fungal species.

Mating disruption of Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) by playback of vibrational signals in vineyard trellis

USDA-ARS?s Scientific Manuscript database

BACKGROUND: Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), is an important vector of the bacterium Xylella fastidiosa, the causal agent of Pierce’s disease of grapevine. Area-wide applications of neonicotinoid insecticides have suppressed GWSS populati...

Playback interference of glassy-winged sharp shooter communication

USDA-ARS?s Scientific Manuscript database

Animal communication is vital to reproduction, particularly for securing a mate. Insects commonly communicate by exchanging vibrational signals that are transmitted through host plants. The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, is an important vector of Xylella fastidiosa, a pl...

Playback of natural vibrational signals in vineyard trellis for mating disruption of glassy-winged sharpshooter

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), is a vector of Xylella fastidiosa, an important bacterial pathogen of several crops in the Americas and Europe. Mating communication of this and many other cicadellid pests involves the exchange of substrate-...

Salmonella enterica Prophage Sequence Profiles Reflect Genome Diversity and Can Be Used for High Discrimination Subtyping.

PubMed

Mottawea, Walid; Duceppe, Marc-Olivier; Dupras, Andrée A; Usongo, Valentine; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Hamel, Jeremie; Kukavica-Ibrulj, Irena; Boyle, Brian; Gill, Alexander; Burnett, Elton; Franz, Eelco; Arya, Gitanjali; Weadge, Joel T; Gruenheid, Samantha; Wiedmann, Martin; Huang, Hongsheng; Daigle, France; Moineau, Sylvain; Bekal, Sadjia; Levesque, Roger C; Goodridge, Lawrence D; Ogunremi, Dele

2018-01-01

Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella . In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S . Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

Glassy-winged sharpshooter oviposition effects on foliar grapevine and red-tipped photinia terpenoid levels

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), is an important vector of Xylella fastidiosa, the bacterium that causes Pierce's disease of grapevine and is a threat to grape production throughout the United States. Female GWSS deposit egg masses be...

Effects of nymphal diet and adult feeding on allocation of resources to glassy-winged sharpshooter egg production

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter is an invasive insect capable of transmitting the bacterial pathogen Xylella fastidiosa. Pre-oviposition periods of laboratory reared glassy-winged sharpshooters are variable. Here, two questions were addressed: does nymphal diet affect pre-oviposition period and how d...

From rags to riches: insights from the first genomic sequence of a plant pathogenic bacterium

PubMed Central

Keen, Noel T; Korsi Dumenyo, C; Yang, Ching-Hong; Cooksey, Donald A

2000-01-01

The recently published genomic sequence of Xylella fastidiosa is the first for a free-living plant pathogen and provides clues to mechanisms of pathogenesis and survival in insect vectors. The sequence data should lead to improved control of this pathogen. PMID:11178244

Modeling deployment of Pierce’s disease resistant grapevines

USDA-ARS?s Scientific Manuscript database

Deployment of Pierce’s disease resistant grapevines is a key solution to mitigating economic losses caused by Xylella fastidiosa. While Pierce’s disease resistant grapevines under development display mild symptoms and have lower bacterial populations than susceptible varieties, all appear to remain ...

Design of a candidate vibrational signal for mating disruption against the glassy-winged sharpshooter, Homalodisca Vitripennis

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, is an important pest of grapevines due to its ability to transmit Xylella fastidiosa, the causal agent of Pierce’s disease. GWSS mating communication is based on vibrational signals; therefore, vibrational mating disruption could be an ...

Propagation of Homalodisca Coagulata Virus-01 via Homalodisca Vitripennis cell culture

USDA-ARS?s Scientific Manuscript database

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These insects are the predominant vectors of Xylella fastidiosa, a xylem-limited bacterium that is the causal agent of Pierce's disease (PD) of grapevin...

Control of Pierce's Disease by Phage

PubMed Central

Das, Mayukh; Bhowmick, Tushar Suvra; Ahern, Stephen J.; Young, Ry; Gonzalez, Carlos F.

2015-01-01

Pierce’s Disease (PD) of grapevines, caused by Xylella fastidiosa subsp. fastidiosa (Xf), is a limiting factor in the cultivation of grapevines in the US. There are presently no effective control methods to prevent or treat PD. The therapeutic and prophylactic efficacy of a phage cocktail composed of four virulent (lytic) phages was evaluated for control of PD. Xf levels in grapevines were significantly reduced in therapeutically or prophylactically treated grapevines. PD symptoms ceased to progress one week post-therapeutic treatment and symptoms were not observed in prophylactically treated grapevines. Cocktail phage levels increased in grapevines in the presence of the host. No in planta phage-resistant Xf isolates were obtained. Moreover, Xf mutants selected for phage resistance in vitro did not cause PD symptoms. Our results indicate that phages have great potential for biocontrol of PD and other economically important diseases caused by Xylella. PMID:26107261

Prophage-mediated defense against viral attack and viral counter-defense

PubMed Central

Dedrick, Rebekah M.; Jacobs-Sera, Deborah; Guerrero Bustamante, Carlos A.; Garlena, Rebecca A.; Mavrich, Travis N.; Pope, Welkin H.; Reyes, Juan C Cervantes; Russell, Daniel A.; Adair, Tamarah; Alvey, Richard; Bonilla, J. Alfred; Bricker, Jerald S.; Brown, Bryony R.; Byrnes, Deanna; Cresawn, Steven G.; Davis, William B.; Dickson, Leon A.; Edgington, Nicholas P.; Findley, Ann M.; Golebiewska, Urszula; Grose, Julianne H.; Hayes, Cory F.; Hughes, Lee E.; Hutchison, Keith W.; Isern, Sharon; Johnson, Allison A.; Kenna, Margaret A.; Klyczek, Karen K.; Mageeney, Catherine M.; Michael, Scott F.; Molloy, Sally D.; Montgomery, Matthew T.; Neitzel, James; Page, Shallee T.; Pizzorno, Marie C.; Poxleitner, Marianne K.; Rinehart, Claire A.; Robinson, Courtney J.; Rubin, Michael R.; Teyim, Joseph N.; Vazquez, Edwin; Ware, Vassie C.; Washington, Jacqueline; Hatfull, Graham F.

2017-01-01

Temperate phages are common and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses infecting mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity, and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages reveals at least five distinct prophage-expressed viral defense systems that interfere with infection of lytic and temperate phages that are either closely-related (homotypic defense) or unrelated (heterotypic defense). Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defense systems include a single-subunit restriction system, a heterotypic exclusion system, and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival, and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, that acts as a highly effective counter-defense system. Prophage-mediated viral defense offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defense promotes phage co-evolution. PMID:28067906

Study the taxonomy of Xylella based on whole genome sequences

USDA-ARS?s Scientific Manuscript database

Members of the genus Xylella cause diseases on many economically important crops in the Americas, including Pierce's disease (PD) of grapevine in U.S., and citrus variegated chlorosis (CVC) disease in Brazil. In the past decade, Xylella-caused diseases from outside the Americas, such as pear leaf sc...

Management of almond leaf scorch disease: long term data on yield, tree vitality, and disease progress

USDA-ARS?s Scientific Manuscript database

Almond leaf scorch (ALS) disease has been a chronic problem for California almond growers. This disease is caused by the bacterial pathogen Xylella fastidiosa and is transmitted by xylem-feeding insects. Previous research suggested that retaining, rather than roguing, ALS-affected trees may be more ...

Induction of prophage lambda by chlorinated organics: Detection of some single-species/single-site carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeMarini, D.M.; Brooks, H.G.

1992-01-01

Twenty-eight chlorinated organic compounds were evaluated for their ability to induce DNA damage using the Microscreen prophage-induction assay in Escherichia coli. Comparison of the performance characteristics of the prophage-induction and Salmonella assays to rodent carcinogenicity assays showed that the prophage-induction assay had a somewhat higher specificity than did the Salmonella assay (70% vs. 50%); sensitivity, concordance, and positive and negative predictivity were similar for the two microbial assays. The Microscreen prophage-induction assay failed to detect eight carcinogens, perhaps due to toxicity or other unknown factors; five of these eight carcinogens were detected by the Salmonella assay. However, the prophage-induction assaymore » did detect six carcinogens that were not detected by the Salmonella assay, and five of these were single-species, single-site carcinogens, mostly mouse liver carcinogens. Some of these carcinogens, such as the chloroethanes, produce free radicals, which may be the basis for their carcinogenicity and ability to induce prophage. The prophage-induction (or other SOS) assay may be useful in identifying some genotoxic chlorinated carcinogens that induce DNA damage that do not revert the standard Salmonella tester strains.« less

EPG waveforms of blue green sharpshooter: impedance and voltage level effects on stylet probing

USDA-ARS?s Scientific Manuscript database

Blue-green sharpshooter (BGSS), Graphocephala atropunctata, is a native California vector of Xylella fastidiosa (Xf) a foregut-borne bacterium that is the causal agent of Pierce’s disease in grapevines. A 3rd-generation, AC-DC electropenetrograph (EPG) was used to record probing behaviors of adult B...

EPG waveform library for Graphocephala atropunctata (Hemiptera: Cicadellidae): Effect of input resistor and voltage levels on waveform appearance and probing behaviors

USDA-ARS?s Scientific Manuscript database

Graphocephala atropunctata is a vector of Xylella fastidiosa (Xf), the causal agent of Pierce’s disease of grapevines. A 3rd-generation, AC-DC electropenetrograph (EPG) was used to record stylet probing and ingestion behaviors of adult G. atropunctata on healthy grapevines. This study presents a com...

Optimizing EPG settings to record blue-green sharpshooter X waves for future studies of grape host plant resistance to Xf inoculation

USDA-ARS?s Scientific Manuscript database

The long-term goal of the research reported in this review is to develop methodology for assessment of grapevine resistant to sharpshooter inoculation of Xylella fastidiosa(Xf)into healthy grapevines, thereby preventing Xf infection. Such a trait would be quite different from the more common mechani...

Metabolic Profiling of Xylem Sap from Pierce’s Disease Resistant and Susceptible Grapevines

USDA-ARS?s Scientific Manuscript database

Pierce’s Disease (PD) of grapevines is caused by a gram-negative, xylem-limited bacterium Xylella fastidiosa (Xf). All Vitis vinifera-based cultivars are highly susceptible to Xf infection. However, some grape species from the southern United States such as V. arizonica, V. Shuttleworthii, and Musca...

Prophage Integrase Typing Is a Useful Indicator of Genomic Diversity in Salmonella enterica

PubMed Central

Colavecchio, Anna; D’Souza, Yasmin; Tompkins, Elizabeth; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Kukavica-Ibrulj, Irena; Boyle, Brian; Bekal, Sadjia; Tamber, Sandeep; Levesque, Roger C.; Goodridge, Lawrence D.

2017-01-01

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S. Enteritidis, and 18 integrase genes in S. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S. Enteritidis, and 9 integrase genes in S. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S. Enteritidis and S. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23

Prophage Integrase Typing Is a Useful Indicator of Genomic Diversity in Salmonella enterica.

PubMed

Colavecchio, Anna; D'Souza, Yasmin; Tompkins, Elizabeth; Jeukens, Julie; Freschi, Luca; Emond-Rheault, Jean-Guillaume; Kukavica-Ibrulj, Irena; Boyle, Brian; Bekal, Sadjia; Tamber, Sandeep; Levesque, Roger C; Goodridge, Lawrence D

2017-01-01

Salmonella enterica is a bacterial species that is a major cause of illness in humans and food-producing animals. S. enterica exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars that have been identified. The development of methods to assess genome diversity in S. enterica will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate S. enterica genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of S. enterica were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated Salmonella serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49 S. enterica genomes (79 integrase genes in the food-associated Salmonella isolates, 50 integrase genes in S . Enteritidis, and 18 integrase genes in S . Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49 S. enterica genomes (44 integrase genes in the food-associated Salmonella isolates, 21 integrase genes in S . Enteritidis, and 9 integrase genes in S . Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of S . Enteritidis and S . Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23

76 FR 8603 - Citrus Seed Imports; Citrus Greening and Citrus Variegated Chlorosis

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-15

... strain of the bacterium Xylella fastidiosa, CVC causes severe chlorosis between veins on the leaves of...\\ ARS researchers did note, however, that the bacterium causing HLB remained at a very low titer in... these 769 seedlings tested positive for the disease. However, titer levels of the bacterium were low...

Identification of genes associated with prophage-like gene transfer agents in the pathogenic intestinal spirochaetes Brachyspira hyodysenteriae, Brachyspira pilosicoli and Brachyspira intermedia.

PubMed

Motro, Yair; La, Tom; Bellgard, Matthew I; Dunn, David S; Phillips, Nyree D; Hampson, David J

2009-03-02

VSH-1 is an unusual prophage-like gene transfer agent (GTA) that has been described in the intestinal spirochaete Brachyspira hyodysenteriae. The GTA does not self-propagate, but it assembles into a virus-like particle and transfers random 7.5kb fragments of host DNA to other B. hyodysenteriae cells. To date the GTA VSH-1 has only been analysed in B. hyodysenteriae strain B204, in which 11 late function genes encoding prophage capsid, tail and lysis elements have been described. The aim of the current study was to look for these 11 genes in the near-complete genomes of B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. intermedia HB60. All 11 genes were found in the three new strains. The GTA genes in WA1 and 95/1000 were contiguous, whilst some of those in HB60 were not-although in all three strains some gene rearrangements were present. A new predicted open reading frame with potential functional importance was found in a consistent position associated with all four GTAs, located between the genes for head protein Hvp24 and tail protein Hvp53, overlapping with the hvp24 sequence. Differences in the nucleotide and predicted amino acid sequences of the GTA genes in the spirochaete strains were consistent with the overall genetic distances between the strains. Hence the GTAs in the two B. hyodysenteriae strains were considered to be strain specific variants, and were designated GTA/Bh-B204 and GTA/Bh-WA1 respectively. The GTAs in the strains of B. intermedia and B. pilosicoli were designated GTA/Bint-HB60 and GTA/Bp-95/1000 respectively. Further work is required to determine the extent to which these GTAs can transfer host genes between different Brachyspira species and strains.

Antibiotics in Feed Induce Prophages in Swine Fecal Microbiomes

PubMed Central

Allen, Heather K.; Looft, Torey; Bayles, Darrell O.; Humphrey, Samuel; Levine, Uri Y.; Alt, David; Stanton, Thaddeus B.

2011-01-01

ABSTRACT Antibiotics are a cost-effective tool for improving feed efficiency and preventing disease in agricultural animals, but the full scope of their collateral effects is not understood. Antibiotics have been shown to mediate gene transfer by inducing prophages in certain bacterial strains; therefore, one collateral effect could be prophage induction in the gut microbiome at large. Here we used metagenomics to evaluate the effect of two antibiotics in feed (carbadox and ASP250 [chlortetracycline, sulfamethazine, and penicillin]) on swine intestinal phage metagenomes (viromes). We also monitored the bacterial communities using 16S rRNA gene sequencing. ASP250, but not carbadox, caused significant population shifts in both the phage and bacterial communities. Antibiotic resistance genes, such as multidrug resistance efflux pumps, were identified in the viromes, but in-feed antibiotics caused no significant changes in their abundance. The abundance of phage integrase-encoding genes was significantly increased in the viromes of medicated swine over that in the viromes of nonmedicated swine, demonstrating the induction of prophages with antibiotic treatment. Phage-bacterium population dynamics were also examined. We observed a decrease in the relative abundance of Streptococcus bacteria (prey) when Streptococcus phages (predators) were abundant, supporting the “kill-the-winner” ecological model of population dynamics in the swine fecal microbiome. The data show that gut ecosystem dynamics are influenced by phages and that prophage induction is a collateral effect of in-feed antibiotics. PMID:22128350

Bacterial leaf scorch distribution and isothermal lines (PROJECT NC-EM-08-02)

Treesearch

Gerard C. Adams; Mursel Catall; James Walla; Ann B. Gould

2013-01-01

Bacterial leaf scorch (BLS) of shade trees is the common name for a disease caused by Xylella fastidiosa, a xylem-inhabiting bacterium that has fastidious nutritional requirements and is difficult to culture or verify by culturing. Forest trees including oak, sycamore, elm, planetree, sweetgum, mulberry and maple are species susceptible to ...

A Type 3 prophage of “Candidatus Liberibacter asiaticus” carrying a restriction-modification system

USDA-ARS?s Scientific Manuscript database

Prophages, the lysogenic form of bacterial phages, are important genetic entities of “Candidatus Liberibacter asiaticus” (CLas), a non-culturable alfa-proteobacterium associated with citrus Huanglongbing (HLB). Two different CLas prophages are currently known, SC1 (Type 1) that has a lytic cycle and...

Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions

PubMed Central

Zeaki, Nikoleta; Budi Susilo, Yusak; Pregiel, Anna; Rådström, Peter; Schelin, Jenny

2015-01-01

The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards. PMID:26690218

Bacterial Leaf Scorch of Amenity Trees a Wide-Spread Problem of Economic Significance to the Urban Forest

Treesearch

James Lashomb; Alan Iskra; Ann Brooks Gould; George Hamilton

2003-01-01

Bacterial leaf scorch (BLS) of amenity trees is caused by the bacterium Xylella fastidiosa, a xylem-limited pathogen that causes water stress resulting in leaf scorch, decline, and eventual death of affected trees. Recent surveys indicate that BLS is widespread throughout the eastern half of the United States. In New Jersey, BLS primarily affects red and pin oaks...

Genome Sequence of Streptomyces wadayamensis Strain A23, an Endophytic Actinobacterium from Citrus reticulata

PubMed Central

Tormet Gonzalez, Gabriela D.; Samborsky, Markyian; Marcon, Joelma; Araujo, Welington L.; de Azevedo, João Lucio

2014-01-01

The actinobacterium Streptomyces wadayamensis A23 is an endophyte of Citrus reticulata that produces the antimycin and mannopeptimycin antibiotics, among others. The strain has the capability to inhibit Xylella fastidiosa growth. The draft genome of S. wadayamensis A23 has ~7.0 Mb and 6,006 protein-coding sequences, with a 73.5% G+C content. PMID:24994795

Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Propage in B. pseudomallei 1026b

DTIC Science & Technology

2004-06-01

identification of several new virulence gene candidates. In particular, K96243 harbors multiple genomic islands with relatively low GC contents, suggesting...coli, Streptococcus pyogenes, Staphylococcus aureus, S. enterica, and Xylella fastidiosa (11, 16, 17). The genomic sequencing results for multiple... virulence genes by subtractive hybridization: identifica- tion of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant

Prophage-mediated dynamics of 'Candidatus Liberibacter asiaticus' populations, the destructive bacterial pathogens of citrus huanglongbing.

PubMed

Zhou, Lijuan; Powell, Charles A; Li, Wenbin; Irey, Mike; Duan, Yongping

2013-01-01

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus' (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.

X-FIDO: An Effective Application for Detecting Olive Quick Decline Syndrome with Deep Learning and Data Fusion.

PubMed

Cruz, Albert C; Luvisi, Andrea; De Bellis, Luigi; Ampatzidis, Yiannis

2017-01-01

We have developed a vision-based program to detect symptoms of Olive Quick Decline Syndrome (OQDS) on leaves of Olea europaea L. infected by Xylella fastidiosa , named X-FIDO ( Xylella FastIdiosa Detector for O. europaea L.). Previous work predicted disease from leaf images with deep learning but required a vast amount of data which was obtained via crowd sourcing such as the PlantVillage project. This approach has limited applicability when samples need to be tested with traditional methods (i.e., PCR) to avoid incorrect training input or for quarantine pests which manipulation is restricted. In this paper, we demonstrate that transfer learning can be leveraged when it is not possible to collect thousands of new leaf images. Transfer learning is the re-application of an already trained deep learner to a new problem. We present a novel algorithm for fusing data at different levels of abstraction to improve performance of the system. The algorithm discovers low-level features from raw data to automatically detect veins and colors that lead to symptomatic leaves. The experiment included images of 100 healthy leaves, 99 X. fastidiosa -positive leaves and 100 X. fastidiosa -negative leaves with symptoms related to other stress factors (i.e., abiotic factors such as water stress or others diseases). The program detects OQDS with a true positive rate of 98.60 ± 1.47% in testing, showing great potential for image analysis for this disease. Results were obtained with a convolutional neural network trained with the stochastic gradient descent method, and ten trials with a 75/25 split of training and testing data. This work shows potential for massive screening of plants with reduced diagnosis time and cost.

Prophage Induction Is Enhanced and Required for Renal Disease and Lethality in an EHEC Mouse Model

PubMed Central

Reynolds, Jared L.; Alteri, Christopher J.; Skinner, Katherine G.; Friedman, Jonathan H.; Eaton, Kathryn A.; Friedman, David I.

2013-01-01

Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage. Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity in the intestine increases Stx2 production. PMID:23555250

Prophage-Mediated Dynamics of ‘Candidatus Liberibacter asiaticus’ Populations, the Destructive Bacterial Pathogens of Citrus Huanglongbing

PubMed Central

Zhou, Lijuan; Powell, Charles A.; Li, Wenbin; Irey, Mike; Duan, Yongping

2013-01-01

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus’ (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease

Host structural carbohydrate induces vector transmission of a bacterial plant pathogen.

PubMed

Killiny, Nabil; Almeida, Rodrigo P P

2009-12-29

Many insect-borne pathogens have complex life histories because they must colonize both hosts and vectors for successful dissemination. In addition, the transition from host to vector environments may require changes in gene expression before the pathogen's departure from the host. Xylella fastidiosa is a xylem-limited plant-pathogenic bacterium transmitted by leafhopper vectors that causes diseases in a number of economically important plants. We hypothesized that factors of host origin, such as plant structural polysaccharides, are important in regulating X. fastidiosa gene expression and mediating vector transmission of this pathogen. The addition of pectin and glucan to a simple defined medium resulted in dramatic changes in X. fastidiosa's phenotype and gene-expression profile. Cells grown in the presence of pectin became more adhesive than in other media tested. In addition, the presence of pectin and glucan in media resulted in significant changes in the expression of several genes previously identified as important for X. fastidiosa's pathogenicity in plants. Furthermore, vector transmission of X. fastidiosa was induced in the presence of both polysaccharides. Our data show that host structural polysaccharides mediate gene regulation in X. fastidiosa, which results in phenotypic changes required for vector transmission. A better understanding of how vector-borne pathogens transition from host to vector, and vice versa, may lead to previously undiscovered disease-control strategies.

As Clear as Mud? Determining the Diversity and Prevalence of Prophages in the Draft Genomes of Estuarine Isolates of Clostridium difficile

PubMed Central

Hargreaves, Katherine R.; Otieno, James R.; Thanki, Anisha; Blades, Matthew J.; Millard, Andrew D.; Browne, Hilary P.; Lawley, Trevor D.; Clokie, Martha R.J.

2015-01-01

The bacterium Clostridium difficile is a significant cause of nosocomial infections worldwide. The pathogenic success of this organism can be attributed to its flexible genome which is characterized by the exchange of mobile genetic elements, and by ongoing genome evolution. Despite its pathogenic status, C. difficile can also be carried asymptomatically, and has been isolated from natural environments such as water and sediments where multiple strain types (ribotypes) are found in close proximity. These include ribotypes which are associated with disease, as well as those that are less commonly isolated from patients. Little is known about the genomic content of strains in such reservoirs in the natural environment. In this study, draft genomes have been generated for 13 C. difficile isolates from estuarine sediments including clinically relevant and environmental associated types. To identify the genetic diversity within this strain collection, whole-genome comparisons were performed using the assemblies. The strains are highly genetically diverse with regards to the C. difficile “mobilome,” which includes transposons and prophage elements. We identified a novel transposon-like element in two R078 isolates. Multiple, related and unrelated, prophages were detected in isolates across ribotype groups, including two novel prophage elements and those related to the transducing phage φC2. The susceptibility of these isolates to lytic phage infection was tested using a panel of characterized phages found from the same locality. In conclusion, estuarine sediments are a source of genetically diverse C. difficile strains with a complex network of prophages, which could contribute to the emergence of new strains in clinics. PMID:26019165

Gene expression analysis of six GC-rich Gram-negative phytopathogens.

PubMed

Fu, Qing-Shan; Li, Feng; Chen, Ling-Ling

2005-07-01

Predicted highly expressed (PHX) genes are comparatively analyzed for six GC-rich Gram-negative phytopathogens, i.e., Ralstonia solanacearum, Agrobacterium tumefaciens, Xanthomonas campestris pv. campestris (Xcc), Xanthomonas axonopodis pv. citri (Xac), Pseudomonas syringae pv. tomato, and Xylella fastidiosa. Enzymes involved in energy metabolism, such as ATP synthase, and genes involved in TCA cycle, are PHX in most bacteria except X. fastidiosa, which prefers an anaerobic environment. Most pathogenicity-related factors, including flagellar proteins and some outer membrane proteins, are PHX, except that flagellar proteins are missing in X. fastidiosa which is spread by insects and does not need to move during invasion. Although type III secretion system apparatus are homologous to flagellar proteins, none of them is PHX, which support the viewpoint that the two types of genes have evolved independently. Furthermore, it is revealed that some biosynthesis-related enzymes are highly expressed in certain bacteria. The PHX genes may provide potential drug targets for the design of new bactericide.

Prophagic DNA Fragments in Streptococcus agalactiae Strains and Association with Neonatal Meningitis

PubMed Central

van der Mee-Marquet, Nathalie; Domelier, Anne-Sophie; Mereghetti, Laurent; Lanotte, Philippe; Rosenau, Agnès; van Leeuwen, Willem; Quentin, Roland

2006-01-01

We identified—by randomly amplified polymorphic DNA (RAPD) analysis at the population level followed by DNA differential display, cloning, and sequencing—three prophage DNA fragments (F5, F7, and F10) in Streptococcus agalactiae that displayed significant sequence similarity to the DNA of S. agalactiae and Streptococcus pyogenes. The F5 sequence aligned with a prophagic gene encoding the large subunit of a terminase, F7 aligned with a phage-associated cell wall hydrolase and a phage-associated lysin, and F10 aligned with a transcriptional regulator (ArpU family) and a phage-associated endonuclease. We first determined the prevalence of F5, F7, and F10 by PCR in a collection of 109 strains isolated in the 1980s and divided into two populations: one with a high risk of causing meningitis (HR group) and the other with a lower risk of causing meningitis (LR group). These fragments were significantly more prevalent in the HR group than in the LR group (P < 0.001). Our findings suggest that lysogeny has increased the ability of some S. agalactiae strains to invade the neonatal brain endothelium. We then determined the prevalence of F5, F7, and F10 by PCR in a collection of 40 strains recently isolated from neonatal meningitis cases for comparison with the cerebrospinal fluid (CSF) strains isolated in the 1980s. The prevalence of the three prophage DNA fragments was similar in these two populations isolated 15 years apart. We suggest that the prophage DNA fragments identified have remained stable in many CSF S. agalactiae strains, possibly due to their importance in virulence or fitness. PMID:16517893

Identification of Xylella fastidiosa antivirulence genes: hemagglutinin adhesins contribute a biofilm maturation to X. fastidios and colonization and attenuate virulence.

PubMed

Guilhabert, Magalie R; Kirkpatrick, Bruce C

2005-08-01

Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels.

As Clear as Mud? Determining the Diversity and Prevalence of Prophages in the Draft Genomes of Estuarine Isolates of Clostridium difficile.

PubMed

Hargreaves, Katherine R; Otieno, James R; Thanki, Anisha; Blades, Matthew J; Millard, Andrew D; Browne, Hilary P; Lawley, Trevor D; Clokie, Martha R J

2015-05-27

The bacterium Clostridium difficile is a significant cause of nosocomial infections worldwide. The pathogenic success of this organism can be attributed to its flexible genome which is characterized by the exchange of mobile genetic elements, and by ongoing genome evolution. Despite its pathogenic status, C. difficile can also be carried asymptomatically, and has been isolated from natural environments such as water and sediments where multiple strain types (ribotypes) are found in close proximity. These include ribotypes which are associated with disease, as well as those that are less commonly isolated from patients. Little is known about the genomic content of strains in such reservoirs in the natural environment. In this study, draft genomes have been generated for 13 C. difficile isolates from estuarine sediments including clinically relevant and environmental associated types. To identify the genetic diversity within this strain collection, whole-genome comparisons were performed using the assemblies. The strains are highly genetically diverse with regards to the C. difficile "mobilome," which includes transposons and prophage elements. We identified a novel transposon-like element in two R078 isolates. Multiple, related and unrelated, prophages were detected in isolates across ribotype groups, including two novel prophage elements and those related to the transducing phage φC2. The susceptibility of these isolates to lytic phage infection was tested using a panel of characterized phages found from the same locality. In conclusion, estuarine sediments are a source of genetically diverse C. difficile strains with a complex network of prophages, which could contribute to the emergence of new strains in clinics. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Xylella fastidosa RTX operons: evidence for the evolution of protein mosaics through novel genetic exchanges.

PubMed

Gambetta, Gregory A; Matthews, Mark A; Syvanen, Michael

2018-05-04

Xylella fastidiosa (Xf) is a gram negative bacterium inhabiting the plant vascular system. In most species this bacterium lives as a benign symbiote, but in several agriculturally important plants (e.g. coffee, citrus, grapevine) Xf is pathogenic. Xf has four loci encoding homologues to hemolysin RTX proteins, virulence factors involved in a wide range of plant pathogen interactions. We show that all four genes are expressed during pathogenesis in grapevine. The sequences from these four genes have a complex repetitive structure. At the C-termini, sequence diversity between strains is what would be expected from orthologous genes. However, within strains there is no N-terminal homology, indicating these loci encode RTXs of different functions and/or specificities. More striking is that many of the orthologous loci between strains share this extreme variation at the N-termini. Thus these RTX orthologues are most easily visualized as fusions between the orthologous C-termini and different N-termini. Further, the four genes are found in operons having a peculiar structure with an extensively duplicated module encoding a small protein with homology to the N-terminal region of the full length RTX. Surprisingly, some of these small peptides are most similar not to their corresponding full length RTX, but to the N-termini of RTXs from other Xf strains, and even other remotely related species. These results demonstrate that these genes are expressed in planta during pathogenesis. Their structure suggests extensive evolutionary restructuring through horizontal gene transfers and heterologous recombination mechanisms. The sum of the evidence suggests these repetitive modules are a novel kind of mobile genetic element.

Gene regulation mediates host specificity of a bacterial pathogen.

PubMed

Killiny, Nabil; Almeida, Rodrigo P P

2011-12-01

Many bacterial plant pathogens have a gene-for-gene relationship that determines host specificity. However, there are pathogens such as the xylem-limited bacterium Xylella fastidiosa that do not carry genes considered essential for the gene-for-gene model, such as those coding for a type III secretion system and effector molecules. Nevertheless, X. fastidiosa subspecies are host specific. A comparison of symptom development and host colonization after infection of plants with several mutant strains in two hosts, grapevines and almonds, indicated that X. fastidiosa virulence mechanisms are similar in those plants. Thus, we tested if modification of gene regulation patterns, by affecting the production of a cell-cell signalling molecule (DSF), impacted host specificity in X. fastidiosa. Results show that disruption of the rpfF locus, required for DSF synthesis, in a strain incapable of causing disease in grapevines, leads to symptom development in that host. These data are indicative that the core machinery required for the colonization of grapevines is present in that strain, and that changes in gene regulation alone can lead X. fastidiosa to exploit a novel host. The study of the evolution and mechanisms of host specificity mediated by gene regulation at the genome level could lead to important insights on the emergence of new diseases. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

A novel non prophage(-like) gene-intervening element within gerE that is reconstituted during sporulation in Bacillus cereus ATCC10987.

PubMed

Abe, Kimihiro; Shimizu, Shin-Ya; Tsuda, Shuhei; Sato, Tsutomu

2017-09-12

Gene rearrangement is a widely-shared phenomenon in spore forming bacteria, in which prophage(-like) elements interrupting sporulation-specific genes are excised from the host genome to reconstitute the intact gene. Here, we report a novel class of gene-intervening elements, named gin, inserted in the 225 bp gerE-coding region of the B. cereus ATCC10987 genome, which generates a sporulation-specific rearrangement. gin has no phage-related genes and possesses three site-specific recombinase genes; girA, girB, and girC. We demonstrated that the gerE rearrangement occurs at the middle stage of sporulation, in which site-specific DNA recombination took place within the 9 bp consensus sequence flanking the disrupted gerE segments. Deletion analysis of gin uncovered that GirC and an additional factor, GirX, are responsible for gerE reconstitution. Involvement of GirC and GirX in DNA recombination was confirmed by an in vitro recombination assay. These results broaden the definition of the sporulation-specific gene rearrangement phenomenon: gene-intervening elements are not limited to phage DNA but may include non-viral genetic elements that carry a developmentally-regulated site-specific recombination system.

Polysaccharide Compositions of Intervessel Pit Membranes Contribute to Pierce’s Disease Resistance of Grapevines1[OA

PubMed Central

Sun, Qiang; Greve, L. Carl; Labavitch, John M.

2011-01-01

Symptom development of Pierce’s disease (PD) in grapevine (Vitis vinifera) depends largely on the ability of the bacterium Xylella fastidiosa to use cell wall-degrading enzymes (CWDEs) to break up intervessel pit membranes (PMs) and spread through the vessel system. In this study, an immunohistochemical technique was developed to analyze pectic and hemicellulosic polysaccharides of intervessel PMs. Our results indicate that PMs of grapevine genotypes with different PD resistance differed in the composition and structure of homogalacturonans (HGs) and xyloglucans (XyGs), the potential targets of the pathogen’s CWDEs. The PMs of PD-resistant grapevine genotypes lacked fucosylated XyGs and weakly methyl-esterified HGs (ME-HGs), and contained a small amount of heavily ME-HGs. In contrast, PMs of PD-susceptible genotypes all had substantial amounts of fucosylated XyGs and weakly ME-HGs, but lacked heavily ME-HGs. The intervessel PM integrity and the pathogen’s distribution in Xylella-infected grapevines also showed differences among the genotypes. In pathogen-inoculated, PD-resistant genotypes PM integrity was well maintained and Xylella cells were only found close to the inoculation site. However, in inoculated PD-susceptible genotypes, PMs in the vessels associated with bacteria lost their integrity and the systemic presence of the X. fastidiosa pathogen was confirmed. Our analysis also provided a relatively clear understanding of the process by which intervessel PMs are degraded. All of these observations support the conclusion that weakly ME-HGs and fucosylated XyGs are substrates of the pathogen’s CWDEs and their presence in or absence from PMs may contribute to grapevine’s PD susceptibility. PMID:21343427

A novel prophage identified in strains from Salmonella enterica serovar Enteritidis is a phylogenetic signature of the lineage ST-1974

PubMed Central

D'Alessandro, Bruno; Pérez Escanda, Victoria; Balestrazzi, Lucía; Iriarte, Andrés; Pickard, Derek; Yim, Lucía; Chabalgoity, José Alejandro; Betancor, Laura

2018-01-01

Salmonella enterica serovar Enteritidis is a major agent of foodborne diseases worldwide. In Uruguay, this serovar was almost negligible until the mid 1990s but since then it has become the most prevalent. Previously, we characterized a collection of strains isolated from 1988 to 2005 and found that the two oldest strains were the most genetically divergent. In order to further characterize these strains, we sequenced and annotated eight genomes including those of the two oldest isolates. We report on the identification and characterization of a novel 44 kbp Salmonella prophage found exclusively in these two genomes. Sequence analysis reveals that the prophage is a mosaic, with homologous regions in different Salmonella prophages. It contains 60 coding sequences, including two genes, gogB and sseK3, involved in virulence and modulation of host immune response. Analysis of serovar Enteritidis genomes available in public databases confirmed that this prophage is absent in most of them, with the exception of a group of 154 genomes. All 154 strains carrying this prophage belong to the same sequence type (ST-1974), suggesting that its acquisition occurred in a common ancestor. We tested this by phylogenetic analysis of 203 genomes representative of the intraserovar diversity. The ST-1974 forms a distinctive monophyletic lineage, and the newly described prophage is a phylogenetic signature of this lineage that could be used as a molecular marker. The phylogenetic analysis also shows that the major ST (ST-11) is polyphyletic and might have given rise to almost all other STs, including ST-1974. PMID:29509137

The Impact of Prophage on the Equilibria and Stability of Phage and Host

NASA Astrophysics Data System (ADS)

Yu, Pei; Nadeem, Alina; Wahl, Lindi M.

2017-06-01

In this paper, we present a bacteriophage model that includes prophage, that is, phage genomes that are incorporated into the host cell genome. The general model is described by an 18-dimensional system of ordinary differential equations. This study focuses on asymptotic behaviour of the model, and thus the system is reduced to a simple six-dimensional model, involving uninfected host cells, infected host cells and phage. We use dynamical system theory to explore the dynamic behaviour of the model, studying in particular the impact of prophage on the equilibria and stability of phage and host. We employ bifurcation and stability theory, centre manifold and normal form theory to show that the system has multiple equilibrium solutions which undergo a series of bifurcations, finally leading to oscillating motions. Numerical simulations are presented to illustrate and confirm the analytical predictions. The results of this study indicate that in some parameter regimes, the host cell population may drive the phage to extinction through diversification, that is, if multiple types of host emerge; this prediction holds even if the phage population is likewise diverse. This parameter regime is restricted, however, if infecting phage are able to recombine with prophage sequences in the host cell genome.

Controlling biofilm formation, prophage excision and cell death by rewiring global regulator H‐NS of Escherichia coli

PubMed Central

Hong, Seok Hoon; Wang, Xiaoxue; Wood, Thomas K.

2010-01-01

Summary The global regulator H‐NS of Escherichia coli controls genes related to stress response, biofilm formation and virulence by recognizing curved DNA and by silencing acquired genes. Here, we rewired H‐NS to control biofilm formation using protein engineering; H‐NS variant K57N was obtained that reduces biofilm formation 10‐fold compared with wild‐type H‐NS (wild‐type H‐NS increases biofilm formation whereas H‐NS K57N reduces it). Whole‐transcriptome analysis revealed that H‐NS K57N represses biofilm formation through its interaction with the nucleoid‐associated proteins Cnu and StpA and in the absence of these proteins, H‐NS K57N was unable to reduce biofilm formation. Significantly, H‐NS K57N enhanced the excision of defective prophage Rac while wild‐type H‐NS represses excision, and H‐NS controlled only Rac excision among the nine resident E. coli K‐12 prophages. Rac prophage excision not only led to the change in biofilm formation but also resulted in cell lysis through the expression of toxin HokD. Hence, the H‐NS regulatory system may be evolved through a single‐amino‐acid change in its N‐terminal oligomerization domain to control biofilm formation, prophage excision and apoptosis. PMID:21255333

Locating and Activating Molecular ‘Time Bombs’: Induction of Mycolata Prophages

PubMed Central

Dyson, Zoe A.; Brown, Teagan L.; Farrar, Ben; Doyle, Stephen R.; Tucci, Joseph; Seviour, Robert J.; Petrovski, Steve

2016-01-01

Little is known about the prevalence, functionality and ecological roles of temperate phages for members of the mycolic acid producing bacteria, the Mycolata. While many lytic phages infective for these organisms have been isolated, and assessed for their suitability for use as biological control agents of activated sludge foaming, no studies have investigated how temperate phages might be induced for this purpose. Bioinformatic analysis using the PHAge Search Tool (PHAST) on Mycolata whole genome sequence data in GenBank for members of the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella revealed 83% contained putative prophage DNA sequences. Subsequent prophage inductions using mitomycin C were conducted on 17 Mycolata strains. This led to the isolation and genome characterization of three novel Caudovirales temperate phages, namely GAL1, GMA1, and TPA4, induced from Gordonia alkanivorans, Gordonia malaquae, and Tsukamurella paurometabola, respectively. All possessed highly distinctive dsDNA genome sequences. PMID:27487243

Bacteria causing important diseases of citrus utilise distinct modes of pathogenesis to attack a common host.

PubMed

Vojnov, Adrián Alberto; do Amaral, Alexandre Morais; Dow, John Maxwell; Castagnaro, Atilio Pedro; Marano, Marìa Rosa

2010-06-01

In this review, we summarise the current knowledge on three pathogens that exhibit distinct tissue specificity and modes of pathogenesis in citrus plants. Xanthomonas axonopodis pv. citri causes canker disease and invades the host leaf mesophyll tissue through natural openings and can also survive as an epiphyte. Xylella fastidiosa and Candidatus Liberibacter are vectored by insects and proliferate in the vascular system of the host, either in the phloem (Candidatus Liberibacter) or xylem (X. fastidiosa) causing variegated chlorosis and huanglongbing diseases, respectively. Candidatus Liberibacter can be found within host cells and is thus unique as an intracellular phytopathogenic bacterium. Genome sequence comparisons have identified groups of species-specific genes that may be associated with the particular lifestyle, mode of transmission or symptoms produced by each phytopathogen. In addition, components that are conserved amongst bacteria may have diverse regulatory actions underpinning the different bacterial lifestyles; one example is the divergent role of the Rpf/DSF cell-cell signalling system in X. citri and X. fastidiosa. Biofilm plays a key role in epiphytic fitness and canker development in X. citri and in the symptoms produced by X. fastidiosa. Bacterial aggregation may be associated with vascular occlusion of the xylem vessels and symptomatology of variegated chlorosis.

ArgO145, a Stx2a prophage of a bovine O145:H- STEC strain, is closely related to phages of virulent human strains.

PubMed

Krüger, A; Burgán, J; Friedrich, A W; Rossen, J W A; Lucchesi, P M A

2018-06-01

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H- strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145. Copyright © 2018 Elsevier B.V. All rights reserved.

Stx1 prophage excision in Escherichia coli strain PA20 confers strong curli and biofilm formation by restoring native mlrA

USDA-ARS?s Scientific Manuscript database

Prophage insertions in Escherichia coli O157:H7 mlrA contribute to the low expression of curli fimbriae and biofilm observed in many clinical isolates. Varying levels of CsgD-dependent curli/biofilm expression are restored to strains bearing prophage insertions in mlrA by mutation of regulatory gene...

Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis

PubMed Central

Abe, Kimihiro; Kawano, Yuta; Iwamoto, Keito; Arai, Kenji; Maruyama, Yuki; Eichenberger, Patrick; Sato, Tsutomu

2014-01-01

Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection. PMID:25299644

Xylem structure of four grape varieties and 12 alternative hosts to the xylem-limited bacterium Xylella fastidious

PubMed Central

Chatelet, David S.; Wistrom, Christina M.; Purcell, Alexander H.; Rost, Thomas L.; Matthews, Mark A.

2011-01-01

Background and Aims The bacterium Xylella fastidiosa (Xf), responsible for Pierce's disease (PD) of grapevine, colonizes the xylem conduits of vines, ultimately killing the plant. However, Vitis vinifera grapevine varieties differ in their susceptibility to Xf and numerous other plant species tolerate Xf populations without showing symptoms. The aim of this study was to examine the xylem structure of grapevines with different susceptibilities to Xf infection, as well as the xylem structure of non-grape plant species that support or limit movement of Xf to determine if anatomical differences might explain some of the differences in susceptibility to Xf. Methods Air and paint were introduced into leaves and stems to examine the connectivity between stem and leaves and the length distribution of their vessels. Leaf petiole and stem anatomies were studied to determine the basis for the free or restricted movement of Xf into the plant. Key Results There were no obvious differences in stem or petiole vascular anatomy among the grape varieties examined, nor among the other plant species that would explain differences in resistance to Xf. Among grape varieties, the more tolerant ‘Sylvaner’ had smaller stem vessel diameters and 20 % more parenchyma rays than the other three varieties. Alternative hosts supporting Xf movement had slightly longer open xylem conduits within leaves, and more connection between stem and leaves, when compared with alternative hosts that limit Xf movement. Conclusions Stem–leaf connectivity via open xylem conduits and vessel length is not responsible for differences in PD tolerance among grape varieties, or for limiting bacterial movement in the tolerant plant species. However, it was found that tolerant host plants had narrower vessels and more parenchyma rays, possibly restricting bacterial movement at the level of the vessels. The implications of xylem structure and connectivity for the means and regulation of bacterial movement are

Analysis of SOS-Induced Spontaneous Prophage Induction in Corynebacterium glutamicum at the Single-Cell Level

PubMed Central

Nanda, Arun M.; Heyer, Antonia; Krämer, Christina; Grünberger, Alexander; Kohlheyer, Dietrich

2014-01-01

The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1 to -3). Recently, it was shown that the large lysogenic prophage CGP3 (∼187 kbp) is excised spontaneously in a small number of cells. In this study, we provide evidence that a spontaneously induced SOS response is partly responsible for the observed spontaneous CGP3 induction. Whereas previous studies focused mainly on the induction of prophages at the population level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (Pint2 and Plysin) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A PrecA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (∼0.2%) exhibited a spontaneous induction of the SOS response. Correlation of PrecA to the activity of downstream SOS genes (PdivS and PrecN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of PrecA and CGP3 promoter fusions displayed a positive correlation at the single-cell level (ρ = 0.44 to 0.77). Furthermore, analysis of the PrecA-eyfp/Pint2-e2-crimson strain during growth revealed the highest percentage of spontaneous PrecA and Pint2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages. PMID:24163339

RS1 Satellite Phage Promotes Diversity of Toxigenic Vibrio cholerae by Driving CTX Prophage Loss and Elimination of Lysogenic Immunity

PubMed Central

Kamruzzaman, M.; Robins, William Paul; Bari, S. M. Nayeemul; Nahar, Shamsun; Mekalanos, John J.

2014-01-01

In El Tor biotype strains of toxigenic Vibrio cholerae, the CTXϕ prophage often resides adjacent to a chromosomally integrated satellite phage genome, RS1, which produces RS1ϕ particles by using CTX prophage-encoded morphogenesis proteins. RS1 encodes RstC, an antirepressor against the CTXϕ repressor RstR, which cooperates with the host-encoded LexA protein to maintain CTXϕ lysogeny. We found that superinfection of toxigenic El Tor strains with RS1ϕ, followed by inoculation of the transductants into the adult rabbit intestine, caused elimination of the resident CTX prophage-producing nontoxigenic derivatives at a high frequency. Further studies using recA deletion mutants and a cloned rstC gene showed that the excision event was recA dependent and that introduction of additional copies of the cloned rstC gene instead of infection with RS1ϕ was sufficient to enhance CTXϕ elimination. Our data suggest that once it is excised from the chromosome, the elimination of CTX prophage from host cells is driven by the inability to reestablish CTXϕ lysogeny while RstC is overexpressed. However, with eventual loss of the additional copies of rstC, the nontoxigenic derivatives can act as precursors of new toxigenic strains by acquiring the CTX prophage either through reinfection with CTXϕ or by chitin-induced transformation. These results provide new insights into the role of RS1ϕ in V. cholerae evolution and the emergence of highly pathogenic clones, such as the variant strains associated with recent devastating epidemics of cholera in Asia, sub-Saharan Africa, and Haiti. PMID:24935981

Solution NMR structure of hypothetical protein CV_2116 encoded by a viral prophage element in Chromobacterium violaceum.

PubMed

Yang, Yunhuang; Ramelot, Theresa A; Cort, John R; Garcia, Maite; Yee, Adelinda; Arrowsmith, Cheryl H; Kennedy, Michael A

2012-01-01

CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e(-07)) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and (13)C- and (15)N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + β fold containing two anti-parallel β-sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages) due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum.

Interaction between transposable phages: cip locus of prophage D3112, responsible for inhibition of integration and transposition of the related phage B39 of Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerasimov, V.A.; Yanenko, A.S.; Akhverdyan, V.Z.

1986-04-01

Bacteriophage D3112 forms two types of PA01 (D3112) lysogens: those that partially, or completely, limit the growth of the related heteroimmune phage B39. DNA/DNA hybridization has shown that the lysogens of the first type always contain one copy of prophage D3112 (monolysogens), and the lysogens of the second type contain two or more copies of prophage D3112. Limitation of the growth of phage B39 on PA01 (D3112) lysogens is associated with the functioning of the locus of prophage D3112, designated as cip (control of interaction of phages). Using deletion derivatives of plasmid RP4::D3112, the cip locus was mapped at anmore » interval of 1.3-2.45 kb of the D3112 genome. The expression of the cip locus occurs only if the D3112 genome is at the prophage state. The function of the Cip prophage of D3112 exerts an influence on early stages of development of phage B39, decreasing the efficiency of the integration and transposition processes of phage B39.« less

Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen.

PubMed

Hargreaves, Katherine R; Flores, Cesar O; Lawley, Trevor D; Clokie, Martha R J

2014-08-26

Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity. Clostridium difficile is a significant bacterial human pathogen which undergoes continual genome evolution, resulting in the emergence of new virulent strains. Phages are major facilitators of genome evolution in other bacterial species, and we use sequence analysis-based approaches in order to examine whether the CRISPR/Cas system could control these interactions across divergent C. difficile strains. The presence of spacer sequences in prophages that are homologous to phage genomes raises an

The extracytoplasmic function sigma factor SigY is important for efficient maintenance of the Spβ prophage that encodes sublancin in Bacillus subtilis.

PubMed

Mendez, Rebecca; Gutierrez, Alba; Reyes, Jasmin; Márquez-Magaña, Leticia

2012-06-01

Many strains of the soil bacterium Bacillus subtilis are capable of producing and being resistant to the antibiotic sublancin because they harbor the Spβ prophage. This 135 kb viral genome is integrated into the circular DNA chromosome of B. subtilis, and contains genes for the production of and resistance to sublancin. We investigated the role of SigY in sublancin production and resistance, finding that it is important for efficient maintenance of the Spβ prophage. We were unable to detect the prophage in mutants lacking SigY. Additionally, these mutants were no longer able to produce sublancin, were sensitive to killing by this factor, and displayed a delay in sporulation. Wild-type cells with normal SigY activity were found to partially lose the Spβ prophage during growth and early sporulation, suggesting a mechanism for the bistable outcome of sibling cells capable of killing and of being killed. The appropriate regulation of SigY appears to be essential for growth as evidenced by the inability to disrupt the gene for its putative antisigma. Our results confirm a role for SigY in antibiotic production and resistance, as has been found for other members of the extracytoplasmic function sigma factor family in B. subtilis, and shows that this role is achieved by affecting maintenance of the Spβ prophage.

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

PubMed Central

Armentrout, Richard W.; Rutberg, Lars

1971-01-01

A temperature-inducible mutant of temperate Bacillus bacteriophage φ105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in “mock-induced” wild-type φ105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome. PMID:5002012

Interaction of Type IV Toxin/Antitoxin Systems in Cryptic Prophages of Escherichia coli K-12.

PubMed

Wen, Zhongling; Wang, Pengxia; Sun, Chenglong; Guo, Yunxue; Wang, Xiaoxue

2017-03-01

Toxin/antitoxin (TA) systems are widespread in prokaryotic chromosomes and in mobile genetic elements including plasmids and prophages. The first characterized Type IV TA system CbtA/CbeA was found in cryptic prophage CP4-44 in Escherichia coli K-12. Two homologous TA loci of CbtA/CbeA also reside in cryptic prophages of E. coli K-12, YkfI/YafW in CP4-6 and YpjF/YfjZ in CP4-57. In this study, we demonstrated that YkfI and YpjF inhibited cell growth and led to the formation of "lemon-shaped" cells. Prolonged overproduction of YkfI led to the formation of "gourd-shaped" cells and immediate cell lysis. YafW and YfjZ can neutralize the toxicity of YkfI or YpjF. Furthermore, we found that YkfI and YpjF interacted with cell division protein FtsZ in E. coli , but ectopic expression in Pseudomonas and Shewanella did not cause the formation of "lemon-shaped" cells. Moreover, deletion of all of the three toxin genes together decreased resistance to oxidative stress and deletion of the antitoxin genes increased early biofilm formation. Collectively, these results demonstrated that the homologous Type IV TA systems in E. coli may target cell division protein FtsZ in E. coli and may have different physiological functions in E. coli .

Interaction of Type IV Toxin/Antitoxin Systems in Cryptic Prophages of Escherichia coli K-12

PubMed Central

Wen, Zhongling; Wang, Pengxia; Sun, Chenglong; Guo, Yunxue; Wang, Xiaoxue

2017-01-01

Toxin/antitoxin (TA) systems are widespread in prokaryotic chromosomes and in mobile genetic elements including plasmids and prophages. The first characterized Type IV TA system CbtA/CbeA was found in cryptic prophage CP4-44 in Escherichia coli K-12. Two homologous TA loci of CbtA/CbeA also reside in cryptic prophages of E. coli K-12, YkfI/YafW in CP4-6 and YpjF/YfjZ in CP4-57. In this study, we demonstrated that YkfI and YpjF inhibited cell growth and led to the formation of “lemon-shaped” cells. Prolonged overproduction of YkfI led to the formation of “gourd-shaped” cells and immediate cell lysis. YafW and YfjZ can neutralize the toxicity of YkfI or YpjF. Furthermore, we found that YkfI and YpjF interacted with cell division protein FtsZ in E. coli, but ectopic expression in Pseudomonas and Shewanella did not cause the formation of “lemon-shaped” cells. Moreover, deletion of all of the three toxin genes together decreased resistance to oxidative stress and deletion of the antitoxin genes increased early biofilm formation. Collectively, these results demonstrated that the homologous Type IV TA systems in E. coli may target cell division protein FtsZ in E. coli and may have different physiological functions in E. coli. PMID:28257056

CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease

PubMed Central

Buerger, Patrick; Wood-Charlson, Elisha M.; Weynberg, Karen D.; Willis, Bette L.; van Oppen, Madeleine J. H.

2016-01-01

AO1 and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD. PMID:28066391

CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease.

PubMed

Buerger, Patrick; Wood-Charlson, Elisha M; Weynberg, Karen D; Willis, Bette L; van Oppen, Madeleine J H

2016-01-01

and Geitlerinema sp. BBD_1991. We suggest that the presence of virulence genes in potential prophage regions, and the CRISPR-Cas defense systems are evidence of an arms race between the respective cyanobacteria and their bacteriophage predators. The presence of such a defense system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, facilitating the progress of BBD.

Diversification of the function of cell-to-cell signaling in regulation of virulence within plant pathogenic xanthomonads.

PubMed

Dow, Max

2008-05-27

The virulence of plant pathogenic bacteria belonging to the genera Xanthomonas and Xylella depends upon cell-to-cell signaling mediated by the diffusible signal molecule DSF (Diffusible Signaling Factor). Synthesis and perception of the DSF signal require products of the rpf gene cluster. The synthesis of DSF depends on RpfF, whereas the RpfC/RpfG two-component system is implicated in DSF perception and signal transduction. The sensor RpfC acts to negatively regulate synthesis of DSF. In Xanthomonas campestris, mutation of rpfF or rpfC leads to a coordinate down-regulation in synthesis of virulence factors and a reduction in virulence. In contrast, in Xylella fastidiosa, the causal agent of Pierce's disease of grape, mutation of rpfF and rpfC have opposite effects on virulence, with rpfF mutants exhibiting a hypervirulent phenotype. The findings suggest that different xanthomonads have adapted the perception and function of similar types of signaling molecule to fit the specific needs for colonization of different hosts.

Evaluation of endophytic colonization of Citrus sinensis and Catharanthus roseus seedlings by endophytic bacteria.

PubMed

Lacava, Paulo Teixeira; Araújo, Welington Luiz; Azevedo, João Lúcio

2007-02-01

Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome.

PubMed

Yamamoto, Kaneyoshi; Yamanaka, Yuki; Shimada, Tomohiro; Sarkar, Paramita; Yoshida, Myu; Bhardwaj, Neerupma; Watanabe, Hiroki; Taira, Yuki; Chatterji, Dipankar; Ishihama, Akira

2018-01-01

The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α 2 ββ'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, β', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ -defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ -deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome

PubMed Central

Yamamoto, Kaneyoshi; Yamanaka, Yuki; Shimada, Tomohiro; Sarkar, Paramita; Yoshida, Myu; Bhardwaj, Neerupma; Watanabe, Hiroki; Taira, Yuki

2018-01-01

ABSTRACT The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2ββ′ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, β′, but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia

Characterization of Sinorhizobium sp. LM21 Prophages and Virus-Encoded DNA Methyltransferases in the Light of Comparative Genomic Analyses of the Sinorhizobial Virome

PubMed Central

Decewicz, Przemyslaw; Radlinska, Monika; Dziewit, Lukasz

2017-01-01

The genus Sinorhizobium/Ensifer mostly groups nitrogen-fixing bacteria that create root or stem nodules on leguminous plants and transform atmospheric nitrogen into ammonia, which improves the productivity of the plants. Although these biotechnologically-important bacteria are commonly found in various soil environments, little is known about their phages. In this study, the genome of Sinorhizobium sp. LM21 isolated from a heavy-metal-contaminated copper mine in Poland was investigated for the presence of prophages and DNA methyltransferase-encoding genes. In addition to the previously identified temperate phage, ΦLM21, and the phage-plasmid, pLM21S1, the analysis revealed the presence of three prophage regions. Moreover, four novel phage-encoded DNA methyltransferase (MTase) genes were identified and the enzymes were characterized. It was shown that two of the identified viral MTases methylated the same target sequence (GANTC) as cell cycle-regulated methyltransferase (CcrM) of the bacterial host strain, LM21. This discovery was recognized as an example of the evolutionary convergence between enzymes of sinorhizobial viruses and their host, which may play an important role in virus cycle. In the last part of the study, thorough comparative analyses of 31 sinorhizobial (pro)phages (including active sinorhizobial phages and novel putative prophages retrieved and manually re-annotated from Sinorhizobium spp. genomes) were performed. The networking analysis revealed the presence of highly conserved proteins (e.g., holins and endolysins) and a high diversity of viral integrases. The analysis also revealed a large number of viral DNA MTases, whose genes were frequently located within the predicted replication modules of analyzed prophages, which may suggest their important regulatory role. Summarizing, complex analysis of the phage protein similarity network enabled a new insight into overall sinorhizobial virome diversity. PMID:28672885

Commercial Biocides Induce Transfer of Prophage Φ13 from Human Strains of Staphylococcus aureus to Livestock CC398.

PubMed

Tang, Yuanyue; Nielsen, Lene N; Hvitved, Annemette; Haaber, Jakob K; Wirtz, Christiane; Andersen, Paal S; Larsen, Jesper; Wolz, Christiane; Ingmer, Hanne

2017-01-01

Human strains of Staphylococcus aureus commonly carry the bacteriophage ΦSa3 that encodes immune evasion factors. Recently, this prophage has been found in livestock-associated, methicillin resistant S. aureus (MRSA) CC398 strains where it may promote human colonization. Here, we have addressed if exposure to biocidal products induces phage transfer, and find that during co-culture, Φ13 from strain 8325, belonging to ΦSa3 group, is induced and transferred from a human strain to LA-MRSA CC398 when exposed to sub-lethal concentrations of commercial biocides containing hydrogen peroxide. Integration of ΦSa3 in LA-MRSA CC398 occurs at multiple positions and the integration site influences the stability of the prophage. We did not observe integration in hlb encoding β-hemolysin that contains the preferred ΦSa3 attachment site in human strains, and we demonstrate that this is due to allelic variation in CC398 strains that disrupts the phage attachment site, but not the expression of β-hemolysin. Our results show that hydrogen peroxide present in biocidal products stimulate transfer of ΦSa3 from human to LA-MRSA CC398 strains and that in these strains prophage stability depends on the integration site. Knowledge of ΦSa3 transfer and stability between human and livestock strains may lead to new intervention measures directed at reducing human infection by LA-MRSA strains.

A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents

PubMed Central

Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

2016-01-01

Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205–279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics. PMID:27460944

A novel roseobacter phage possesses features of podoviruses, siphoviruses, prophages and gene transfer agents

NASA Astrophysics Data System (ADS)

Zhan, Yuanchao; Huang, Sijun; Voget, Sonja; Simon, Meinhard; Chen, Feng

2016-07-01

Bacteria in the Roseobacter lineage have been studied extensively due to their significant biogeochemical roles in the marine ecosystem. However, our knowledge on bacteriophage which infects the Roseobacter clade is still very limited. Here, we report a new bacteriophage, phage DSS3Φ8, which infects marine roseobacter Ruegeria pomeroyi DSS-3. DSS3Φ8 is a lytic siphovirus. Genomic analysis showed that DSS3Φ8 is most closely related to a group of siphoviruses, CbK-like phages, which infect freshwater bacterium Caulobacter crescentus. DSS3Φ8 contains a smaller capsid and has a reduced genome size (146 kb) compared to the CbK-like phages (205-279 kb). DSS3Φ8 contains the DNA polymerase gene which is closely related to T7-like podoviruses. DSS3Φ8 also contains the integrase and repressor genes, indicating its potential to involve in lysogenic cycle. In addition, four GTA (gene transfer agent) genes were identified in the DSS3Φ8 genome. Genomic analysis suggests that DSS3Φ8 is a highly mosaic phage that inherits the genetic features from siphoviruses, podoviruses, prophages and GTAs. This is the first report of CbK-like phages infecting marine bacteria. We believe phage isolation is still a powerful tool that can lead to discovery of new phages and help interpret the overwhelming unknown sequences in the viral metagenomics.

Commercial Biocides Induce Transfer of Prophage Φ13 from Human Strains of Staphylococcus aureus to Livestock CC398

PubMed Central

Tang, Yuanyue; Nielsen, Lene N.; Hvitved, Annemette; Haaber, Jakob K.; Wirtz, Christiane; Andersen, Paal S.; Larsen, Jesper; Wolz, Christiane; Ingmer, Hanne

2017-01-01

Human strains of Staphylococcus aureus commonly carry the bacteriophage ΦSa3 that encodes immune evasion factors. Recently, this prophage has been found in livestock-associated, methicillin resistant S. aureus (MRSA) CC398 strains where it may promote human colonization. Here, we have addressed if exposure to biocidal products induces phage transfer, and find that during co-culture, Φ13 from strain 8325, belonging to ΦSa3 group, is induced and transferred from a human strain to LA-MRSA CC398 when exposed to sub-lethal concentrations of commercial biocides containing hydrogen peroxide. Integration of ΦSa3 in LA-MRSA CC398 occurs at multiple positions and the integration site influences the stability of the prophage. We did not observe integration in hlb encoding β-hemolysin that contains the preferred ΦSa3 attachment site in human strains, and we demonstrate that this is due to allelic variation in CC398 strains that disrupts the phage attachment site, but not the expression of β-hemolysin. Our results show that hydrogen peroxide present in biocidal products stimulate transfer of ΦSa3 from human to LA-MRSA CC398 strains and that in these strains prophage stability depends on the integration site. Knowledge of ΦSa3 transfer and stability between human and livestock strains may lead to new intervention measures directed at reducing human infection by LA-MRSA strains. PMID:29270158

Compositions and Methods for the Treatment of Pierce's Disease

DOEpatents

Gupta, Goutam

2008-10-07

Chimeric anti-microbial proteins, compositions, and methods for the therapeutic and prophylactic treatment of plant diseases caused by the bacterial pathogen Xylella fastidiosa are provided. The anti-microbial proteins of the invention generally comprise a surface recognition domain polypeptide, capable of binding to a bacterial membrane component, fused to a bacterial lysis domain polypeptide, capable of affecting lysis or rupture of the bacterial membrane, typically via a fused polypeptide linker. In particular, methods and compositions for the treatment or prevention of Pierce's disease of grapevines are provided. Methods for the generation of transgenic Vitus vinefera plants expressing xylem-secreted anti-microbial chimeras are also provided.

comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation, and persistence.

PubMed

Verghese, Bindhu; Lok, Mei; Wen, Jia; Alessandria, Valentina; Chen, Yi; Kathariou, Sophia; Knabel, Stephen

2011-05-01

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as "adaptons," as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

Iron triggers λSo prophage induction and release of extracellular DNA in Shewanella oneidensis MR-1 biofilms.

PubMed

Binnenkade, Lucas; Teichmann, Laura; Thormann, Kai M

2014-09-01

Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H2O2). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H2O2 in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evolution of a zoonotic pathogen: investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing

USDA-ARS?s Scientific Manuscript database

Enterohaemorrhagic E. coli 0157 is a zoonotic pathogen for which colonisation of cattle and virulence in humans is associated with the expression of multiple horizontally acquired genes, the majority present in active or cryptic prophages. Our understanding of the evolution and phylogeny of E. coli ...

Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551.

PubMed

Ahmad, Abdelmonim Ali; Stulberg, Michael J; Huang, Qi

2017-01-01

We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum , indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage's 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes ( pilT, egl, pehC, hrPB, and phcA ), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and

comK Prophage Junction Fragments as Markers for Listeria monocytogenes Genotypes Unique to Individual Meat and Poultry Processing Plants and a Model for Rapid Niche-Specific Adaptation, Biofilm Formation, and Persistence ▿ †

PubMed Central

Verghese, Bindhu; Lok, Mei; Wen, Jia; Alessandria, Valentina; Chen, Yi; Kathariou, Sophia; Knabel, Stephen

2011-01-01

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as “adaptons,” as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety. PMID:21441318

Temporal and spatial scaling of the genetic structure of a vector-borne plant pathogen.

PubMed

Coletta-Filho, Helvécio D; Francisco, Carolina S; Almeida, Rodrigo P P

2014-02-01

The ecology of plant pathogens of perennial crops is affected by the long-lived nature of their immobile hosts. In addition, changes to the genetic structure of pathogen populations may affect disease epidemiology and management practices; examples include local adaptation of more fit genotypes or introduction of novel genotypes from geographically distant areas via human movement of infected plant material or insect vectors. We studied the genetic structure of Xylella fastidiosa populations causing disease in sweet orange plants in Brazil at multiple scales using fast-evolving molecular markers (simple-sequence DNA repeats). Results show that populations of X. fastidiosa were regionally isolated, and that isolation was maintained for populations analyzed a decade apart from each other. However, despite such geographic isolation, local populations present in year 2000 were largely replaced by novel genotypes in 2009 but not as a result of migration. At a smaller spatial scale (individual trees), results suggest that isolates within plants originated from a shared common ancestor. In summary, new insights on the ecology of this economically important plant pathogen were obtained by sampling populations at different spatial scales and two different time points.

Different Expression Patterns of Genes from the Exo-Xis Region of Bacteriophage λ and Shiga Toxin-Converting Bacteriophage Ф24B following Infection or Prophage Induction in Escherichia coli

PubMed Central

Bloch, Sylwia; Nejman-Faleńczyk, Bożena; Dydecka, Aleksandra; Łoś, Joanna M.; Felczykowska, Agnieszka; Węgrzyn, Alicja; Węgrzyn, Grzegorz

2014-01-01

Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide). This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation. PMID:25310402

Initiation of a pan-genomic research project for Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Differences in genomic structure and nucleotide polymorphism among strains form the genetic basis for adaptability of a bacterial species. This can be described by a bacterial pan-genome, which is defined as the full complement of genes in all strains of a species. The pan-genome is composed of a "c...

Abundant and Diverse Clustered Regularly Interspaced Short Palindromic Repeat Spacers in Clostridium difficile Strains and Prophages Target Multiple Phage Types within This Pathogen

PubMed Central

Hargreaves, Katherine R.; Flores, Cesar O.; Lawley, Trevor D.

2014-01-01

ABSTRACT Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity. PMID:25161187

GENOTOXICITY OF 1,3-DICHLOROPROPANE, 2,2-DICHLOROPROPANE, AND L,1-DICHLOROPROPENE IN SALMONELLA AND E. COLI PROPHAGE-INDUCTION ASSAYS

EPA Science Inventory

Genotoxicity of 1,3-Dichloropropane, 2,2-Dichloropropane, and 1,1-Dichloropropene in
Salmonella and E. coli Prophage-Induction Assays

1,3-Dichloropropane (1,3-DCP), 2,2-dichloropropane (2,2-DCP), and 1,1- dichloropropene (I,I-DCP) have been detected in ground water i...

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage.

PubMed

Dey, Arup; Vassallo, Christopher N; Conklin, Austin C; Pathak, Darshankumar T; Troselj, Vera; Wall, Daniel

2016-01-19

mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Sibling Rivalry in Myxococcus xanthus Is Mediated by Kin Recognition and a Polyploid Prophage

PubMed Central

Dey, Arup; Vassallo, Christopher N.; Conklin, Austin C.; Pathak, Darshankumar T.; Troselj, Vera

2016-01-01

siblings, and in part, this is mediated by the TraA cell surface receptor. Surprisingly, we report that TraA recognition can also involve sibling killing. We show that killing originates from a prophage-like element that has apparently hijacked the TraA system to deliver a toxin to kin. We hypothesize that this killing system has imposed selective pressures on kin recognition, which in turn has resulted in TraA polymorphisms and hence many different recognition groups. PMID:26787762

Collagen-Like Proteins in Pathogenic E. coli Strains

PubMed Central

Ghosh, Neelanjana; McKillop, Thomas J.; Jowitt, Thomas A.; Howard, Marjorie; Davies, Heather; Holmes, David F.; Roberts, Ian S.; Bella, Jordi

2012-01-01

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages. PMID:22701585

Persistence of plasmids, cholera toxin genes, and prophage DNA in classical Vibrio cholerae O1.

PubMed

Cook, W L; Wachsmuth, K; Johnson, S R; Birkness, K A; Samadi, A R

1984-07-01

Plasmid profiles, the location of cholera toxin subunit A genes, and the presence of the defective VcA1 prophage genome in classical Vibrio cholerae isolated from patients in Bangladesh in 1982 were compared with those in older classical strains isolated during the sixth pandemic and with those in selected eltor and nontoxigenic O1 isolates. Classical strains typically had two plasmids (21 and 3 megadaltons), eltor strains typically had no plasmids, and nontoxigenic O1 strains had zero to three plasmids. The old and new isolates of classical V. cholerae had two HindIII chromosomal digest fragments containing cholera toxin subunit A genes, whereas the eltor strains from Eastern countries had one fragment. The eltor strains from areas surrounding the Gulf of Mexico also had two subunit A gene fragments, which were smaller and easily distinguished from the classical pattern. All classical strains had 8 to 10 HindIII fragments containing the defective VcA1 prophage genome; none of the Eastern eltor strains had these genes, and the Gulf Coast eltor strains contained a different array of weakly hybridizing genes. These data suggest that the recent isolates of classical cholera in Bangladesh are closely related to the bacterial strain(s) which caused classical cholera during the sixth pandemic. These data do not support hypotheses that either the eltor or the nontoxigenic O1 strains are precursors of the new classical strains.

RNA-seq analysis of prophage induction in multidrug-resistant salmonella enterica serovar typhimurium DT104 following exposure to the agricultural antibiotic carbadox

USDA-ARS?s Scientific Manuscript database

Non-typhoidal Salmonella is a leading cause of U.S. foodborne disease and food-related deaths. Multidrug-resistant (MDR) Salmonella Typhimurium DT104 contains 5 prophages in the genome that may be induced to produce phage under various environmental conditions, including antibiotic exposure. We inve...

φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity.

PubMed

Kvitko, Brian H; Cox, Christopher R; DeShazer, David; Johnson, Shannon L; Voorhees, Kent J; Schweizer, Herbert P

2012-12-07

Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237. We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains. The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.

Early lysis of Lactobacillus helveticus CNRZ 303 in Swiss cheese is not prophage-related.

PubMed

Deutsch, Stéphanie Marie; Neveu, Anthony; Guezenec, Stéphane; Ritzenthaler, Paul; Lortal, Sylvie

2003-03-15

Lactobacillus helveticus is mainly used as starter in Swiss-type cheeses. Often, lysogenic strains are eliminated because of the risk of early lysis and acidification failure due to phage expression. On the other hand, L. helveticus lysis was shown to positively influence cheese proteolysis during ripening. In order to better assess the relationship between lysis and lysogeny, a prophage-cured derivative of L. helveticus CNRZ 303 was isolated (LH 303-G11) and relysogenised (LH 303-G11R), as demonstrated by hybridisation using the whole phage DNA as probe. The growth, lysis in buffered solutions and lytic activities in zymogram using either Micrococcus luteus or L. helveticus as substrate were identical between the mother strain and its cured derivatives. Only morphological differences were observed by scanning electron microscopy: the cells of the cured derivative were shorter in length. The mother strain and its cured and relysogenised derivatives were assayed in triplicate in experimental Swiss cheeses (scale 1:100). No differences were noted during the cheese making: the three strains exhibited identical kinetics of acidification, leading to similar cheeses at day 1 in terms of gross composition and pH. Phages were detected only in the cheeses made with the mother strain and the relysogenised derivative. The lysis of L. helveticus, estimated by viability decrease and release of the intracellular marker D-lactate deshydrogenase, started early before brining and continued during the cold room ripening. No obvious differences of lysis extent were observed. These results demonstrated for the first time that, in the case of LH 303, the extensive lysis observed in cheese is mainly due to autolysin activity and not to prophage induction.

Virulence of plant pathogenic bacteria attenuated by degradation of fatty acid cell-to-cell signaling factors.

PubMed

Newman, Karyn L; Chatterjee, Subhadeep; Ho, Kimberly A; Lindow, Steven E

2008-03-01

Diffusible signal factor (DSF) is a fatty acid signal molecule involved in regulation of virulence in several Xanthomonas species as well as Xylella fastidiosa. In this study, we identified a variety of bacteria that could disrupt DSF-mediated induction of virulence factors in Xanthomonas campestris pv. campestris. While many bacteria had the ability to degrade DSF, several bacterial strains belonging to genera Bacillus, Paenibacillus, Microbacterium, Staphylococcus, and Pseudomonas were identified that were capable of particularly rapid degradation of DSF. The molecular determinants for rapid degradation of DSF in Pseudomonas spp. strain G were elucidated. Random transposon mutants of strain G lacking the ability to degrade DSF were isolated. Cloning and characterization of disrupted genes in these strains revealed that carAB, required for the synthesis of carbamoylphosphate, a precursor for pyrimidine and arginine biosynthesis is required for rapid degradation of DSF in strain G. Complementation of carAB mutants restored both pyrimidine prototrophy and DSF degradation ability of the strain G mutant. An Escherichia coli strain harboring carAB of Pseudomonas spp. strain G degrades DSF more rapidly than the parental strain, and overexpression of carAB in trans increased the ability of Pseudomonas spp. strain G to degrade as compared with the parental strain. Coinoculation of X. campestris pv. campestris with DSF-degrading bacteria into mustard and cabbage leaves reduced disease severity up to twofold compared with plants inoculated only with the pathogen. Likewise, disease incidence and severity in grape stems coinoculated with Xylella fastidiosa and DSF-degrading strains were significantly reduced compared with plants inoculated with the pathogen alone. Coinoculation of grape plants with a carAB mutant of Pseudomonas spp. strain G complemented with carAB in trans reduced disease severity as well or better than the parental strain. These results indicate that

Phage-mediated horizontal gene transfer of both prophage and heterologous DNA by ϕBB-1, a bacteriophage of Borrelia burgdorferi.

PubMed

Eggers, Christian H; Gray, Carlie M; Preisig, Alexander M; Glenn, Danielle M; Pereira, Jessica; Ayers, Ryan W; Alshahrani, Mohammad; Acabbo, Christopher; Becker, Maria R; Bruenn, Kimberly N; Cheung, Timothy; Jendras, Taylor M; Shepley, Aron B; Moeller, John T

2016-12-01

Horizontal gene transfer (HGT) in Borrelia burgdorferi, the Lyme disease agent, is likely mediated by bacteriophage. Studies of the B. burgdorferi phage, ϕBB-1 and its role in HGT have been hindered by the lack of an assay for readily characterizing phage-mediated DNA movement (transduction). Here we describe an in vitro assay in which a clone of B. burgdorferi strain CA-11.2A encoding kanamycin resistance on a ϕBB-1 prophage is co-cultured with different clones encoding gentamicin resistance on a shuttle vector; transduction is monitored by enumerating colonies selected in the presence of both kanamycin and gentamicin. When both clones used in the assay were derived from CA-11.2A, the frequency of transduction was 1.23 × 10 -6 transductants per cell, and could be increased 5-fold by exposing the phage-producing strain to 5% ethanol. Transduction was also demonstrated between the CA-11.2A clone and clones of both high-passage B. burgdorferi strain B31 and low-passage, virulent B. burgdorferi strain 297, although with lower transduction frequencies. The transductant in the 297 background produced phage capable of transducing another B. burgdorferi clone: this is the first experimental demonstration of transduction from a clone of a virulent strain. In addition to prophage DNA, small Escherichia coli-derived shuttle vectors were also transduced between co-cultured B. burgdorferi strains, suggesting both a broad role for the phage in the HGT of heterologous DNA and a potential use of the phage as a molecular tool. These results enhance our understanding of phage-mediated transduction as a mechanism of HGT in the Lyme disease spirochetes. Furthermore, the reagents and techniques developed herein will facilitate future studies of phage-mediated HGT, especially within the tick vector and vertebrate host. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Influence of Xylella fastidiosa cold shock proteins on pathogenesis in grapevine.

USDA-ARS?s Scientific Manuscript database

Cold shock proteins (CSPs), a family of nucleic acid binding proteins are an essential part of microbial adaptation to temperature changes. Bacterial CSPs are often expressed in a temperature-dependent manner, and act as chaperones, facilitating translation at low temperature by stabilizing mRNA. In...

Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551

PubMed Central

Ahmad, Abdelmonim Ali; Stulberg, Michael J.; Huang, Qi

2017-01-01

We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage’s 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and

Physiological Function of Rac Prophage During Biofilm Formation and Regulation of Rac Excision in Escherichia coli K-12

DTIC Science & Technology

2015-11-04

Active prophages such as Gifsy-2 can also give the Salmonella host a competitive advantage by killing competitors and by providing immunity6. In E. coli...to the lack of an active integrase for Qin and CP4-447,31. As oxidative stress is involved in biofilm formation, we tested whether oxidative stress...carbenicillin37. As shown in Fig. 6A, the Δ ttcA strain showed increased metabolic activity compared to that of the wild-type strain without carbenicillin

Escherichia coli O157:H7 Strain EDL933 Harbors Multiple Functional Prophage-Associated Genes Necessary for the Utilization of 5-N-Acetyl-9-O-Acetyl Neuraminic Acid as a Growth Substrate

PubMed Central

Saile, Nadja; Voigt, Anja; Kessler, Sarah; Stressler, Timo; Fischer, Lutz

2016-01-01

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 harbors multiple prophage-associated open reading frames (ORFs) in its genome which are highly homologous to the chromosomal nanS gene. The latter is part of the nanCMS operon, which is present in most E. coli strains and encodes an esterase which is responsible for the monodeacetylation of 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). Whereas one prophage-borne ORF (z1466) has been characterized in previous studies, the functions of the other nanS-homologous ORFs are unknown. In the current study, the nanS-homologous ORFs of EDL933 were initially studied in silico. Due to their homology to the chromosomal nanS gene and their location in prophage genomes, we designated them nanS-p and numbered the different nanS-p alleles consecutively from 1 to 10. The two alleles nanS-p2 and nanS-p4 were selected for production of recombinant proteins, their enzymatic activities were investigated, and differences in their temperature optima were found. Furthermore, a function of these enzymes in substrate utilization could be demonstrated using an E. coli C600ΔnanS mutant in a growth medium with Neu5,9Ac2 as the carbon source and supplementation with the different recombinant NanS-p proteins. Moreover, generation of sequential deletions of all nanS-p alleles in strain EDL933 and subsequent growth experiments demonstrated a gene dose effect on the utilization of Neu5,9Ac2. Since Neu5,9Ac2 is an important component of human and animal gut mucus and since the nutrient availability in the large intestine is limited, we hypothesize that the presence of multiple Neu5,9Ac2 esterases provides them a nutrient supply under certain conditions in the large intestine, even if particular prophages are lost. IMPORTANCE In this study, a group of homologous prophage-borne nanS-p alleles and two of the corresponding enzymes of enterohemorrhagic E. coli (EHEC) O157:H7 strain EDL933 that may be important to provide

Proteomics approach to identify unique xylem sap proteins in Pierce's disease-tolerant Vitis species.

PubMed

Basha, Sheikh M; Mazhar, Hifza; Vasanthaiah, Hemanth K N

2010-03-01

Pierce's disease (PD) is a destructive bacterial disease of grapes caused by Xylella fastidiosa which is xylem-confined. The tolerance level to this disease varies among Vitis species. Our research was aimed at identifying unique xylem sap proteins present in PD-tolerant Vitis species. The results showed wide variation in the xylem sap protein composition, where a set of polypeptides with pI between 4.5 and 4.7 and M(r) of 31 kDa were present in abundant amount in muscadine (Vitis rotundifolia, PD-tolerant), in reduced levels in Florida hybrid bunch (Vitis spp., PD-tolerant) and absent in bunch grapes (Vitis vinifera, PD-susceptible). Liquid chromatography/mass spectrometry/mass spectrometry analysis of these proteins revealed their similarity to beta-1, 3-glucanase, peroxidase, and a subunit of oxygen-evolving enhancer protein 1, which are known to play role in defense and oxygen generation. In addition, the amount of free amino acids and soluble sugars was found to be significantly lower in xylem sap of muscadine genotypes compared to V. vinifera genotypes, indicating that the higher nutritional value of bunch grape sap may be more suitable for Xylella growth. These data suggest that the presence of these unique proteins in xylem sap is vital for PD tolerance in muscadine and Florida hybrid bunch grapes.

Comparative analysis of tandem T7-like promoter containing regions in enterobacterial genomes reveals a novel group of genetic islands | Center for Cancer Research

Cancer.gov

Twelve prophage-like T7 islands have been discovered in pathogenic bacterial genomes. These islands contain two or three tandem T7-like promoters that should be activated when a bacterial cell is infected by bacteriophage T7 or a related phage. The illustration shows genetic maps for four of the islands, Ty2, BS512, E22 and ECA, which are found in the genomes of S. enterica

GENOTOXICITY OF 1,3-DICHLOROPROPANE, 2,2-DICHLOROPROPANE, AND 1,1-DICHLOROPROPENE IN SALMONELLA, THE E. COLI PROPHAGE-INDUCTION ASSAY, AND HUMAN HEPH2 CELLS

EPA Science Inventory

Genotoxicity of 1,3-Dichloropropane, 2,2-Dichloropropane, and 1,1-Dichloropropene in Salmonella, the E. coli Prophage-Induction Assay and Human HepG2 Cells

1,3-Dichloropropane (1,3-DCP), 2,2-dichloropropane (2,2-DCP), and 1,1- dichloropropene ( 1,1- DCP) have been detecte...

Dynamic interactions between prophages induce lysis in Propionibacterium acnes.

PubMed

L Brown, Teagan; Tucci, Joseph; Dyson, Zoe A; Lock, Peter; Adda, Christopher G; Petrovski, Steve

Progress in next-generation sequencing technologies has facilitated investigations into microbial dynamics. An important bacterium in the dairy industry is Propionibacterium freudenreichii, which is exploited to manufacture Swiss cheeses. A healthy culture of these bacteria ensures a consistent cheese with formed 'eyes' and pleasant flavour profile, and the investigation of prophages and their interactions with these bacteria could assist in the maintenance of the standard of this food product. Two bacteriophages, termed PFR1 and PFR2, were chemically induced using mitomycin C from two different dairy strains of P. freudenreichii. Both phages have identical genomes; however, PFR2 was found to contain an insertion sequence, IS204. Host range characterisation showed that PFR1 was able to form plaques on a wild type Propionibacterium acnes strain, whereas PFR2 could not. The lytic plaques observed on P. acnes were a result of PFR1 inducing the lytic cycle of a pseudolysogenic phage in P. acnes. Further investigation revealed that both PFR1 and PFR2 could infect P. acnes but not replicate. This study demonstrates the dynamic interactions between phages, which may alter their lytic capacity under certain conditions. To our knowledge, this is the first report of two phages interacting to kill their host. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Physiological Function of Rac Prophage During Biofilm Formation and Regulation of Rac Excision in Escherichia coli K-12.

PubMed

Liu, Xiaoxiao; Li, Yangmei; Guo, Yunxue; Zeng, Zhenshun; Li, Baiyuan; Wood, Thomas K; Cai, Xingsheng; Wang, Xiaoxue

2015-11-04

Rac or rac-like prophage harbors many genes with important physiological functions, while it remains excision-proficient in several bacterial strains including Escherichia coli, Salmonella spp. and Shigella spp. Here, we found that rac excision is induced during biofilm formation, and the isogenic stain without rac is more motile and forms more biofilms in nutrient-rich medium at early stages in E. coli K-12. Additionally, the presence of rac genes increases cell lysis during biofilm development. In most E. coli strains, rac is integrated into the ttcA gene which encodes a tRNA-thioltransferase. Rac excision in E. coli K-12 leads to a functional change of TtcA, which results in reduced fitness in the presence of carbenicillin. Additionally, we demonstrate that YdaQ (renamed as XisR) is the excisionase of rac in E. coli K-12, and that rac excision is induced by the stationary sigma factor RpoS through inducing xisR expression. Taken together, our results reveal that upon rac integration, not only are new genes introduced into the host, but also there is a functional change in a host enzyme. Hence, rac excision is tightly regulated by host factors to control its stability in the host genome under different stress conditions.

Physiological Function of Rac Prophage During Biofilm Formation and Regulation of Rac Excision in Escherichia coli K-12

PubMed Central

Liu, Xiaoxiao; Li, Yangmei; Guo, Yunxue; Zeng, Zhenshun; Li, Baiyuan; Wood, Thomas K.; Cai, Xingsheng; Wang, Xiaoxue

2015-01-01

Rac or rac-like prophage harbors many genes with important physiological functions, while it remains excision-proficient in several bacterial strains including Escherichia coli, Salmonella spp. and Shigella spp. Here, we found that rac excision is induced during biofilm formation, and the isogenic stain without rac is more motile and forms more biofilms in nutrient-rich medium at early stages in E. coli K-12. Additionally, the presence of rac genes increases cell lysis during biofilm development. In most E. coli strains, rac is integrated into the ttcA gene which encodes a tRNA-thioltransferase. Rac excision in E. coli K-12 leads to a functional change of TtcA, which results in reduced fitness in the presence of carbenicillin. Additionally, we demonstrate that YdaQ (renamed as XisR) is the excisionase of rac in E. coli K-12, and that rac excision is induced by the stationary sigma factor RpoS through inducing xisR expression. Taken together, our results reveal that upon rac integration, not only are new genes introduced into the host, but also there is a functional change in a host enzyme. Hence, rac excision is tightly regulated by host factors to control its stability in the host genome under different stress conditions. PMID:26530864

Sequence variability of Campylobacter temperate bacteriophages

PubMed Central

Clark, Clifford G; Ng, Lai-King

2008-01-01

Background Prophages integrated within the chromosomes of Campylobacter jejuni isolates have been demonstrated very recently. Prior work with Campylobacter temperate bacteriophages, as well as evidence from prophages in other enteric bacteria, suggests these prophages might have a role in the biology and virulence of the organism. However, very little is known about the genetic variability of Campylobacter prophages which, if present, could lead to differential phenotypes in isolates carrying the phages versus those that do not. As a first step in the characterization of C. jejuni prophages, we investigated the distribution of prophage DNA within a C. jejuni population assessed the DNA and protein sequence variability within a subset of the putative prophages found. Results Southern blotting of C. jejuni DNA using probes from genes within the three putative prophages of the C. jejuni sequenced strain RM 1221 demonstrated the presence of at least one prophage gene in a large proportion (27/35) of isolates tested. Of these, 15 were positive for 5 or more of the 7 Campylobacter Mu-like phage 1 (CMLP 1, also designated Campylobacter jejuni integrated element 1, or CJIE 1) genes tested. Twelve of these putative prophages were chosen for further analysis. DNA sequencing of a 9,000 to 11,000 nucleotide region of each prophage demonstrated a close homology with CMLP 1 in both gene order and nucleotide sequence. Structural and sequence variability, including short insertions, deletions, and allele replacements, were found within the prophage genomes, some of which would alter the protein products of the ORFs involved. No insertions of novel genes were detected within the sequenced regions. The 12 prophages and RM 1221 had a % G+C very similar to C. jejuni sequenced strains, as well as promoter regions characteristic of C. jejuni. None of the putative prophages were successfully induced and propagated, so it is not known if they were functional or if they represented remnant

Repression of YdaS Toxin Is Mediated by Transcriptional Repressor RacR in the Cryptic rac Prophage of Escherichia coli K-12.

PubMed

Krishnamurthi, Revathy; Ghosh, Swagatha; Khedkar, Supriya; Seshasayee, Aswin Sai Narain

2017-01-01

Horizontal gene transfer is a major driving force behind the genomic diversity seen in prokaryotes. The cryptic rac prophage in Escherichia coli K-12 carries the gene for a putative transcription factor RacR, whose deletion is lethal. We have shown that the essentiality of racR in E. coli K-12 is attributed to its role in transcriptionally repressing toxin gene(s) called ydaS and ydaT , which are adjacent to and coded divergently to racR . IMPORTANCE Transcription factors in the bacterium E. coli are rarely essential, and when they are essential, they are largely toxin-antitoxin systems. While studying transcription factors encoded in horizontally acquired regions in E. coli , we realized that the protein RacR, a putative transcription factor encoded by a gene on the rac prophage, is an essential protein. Here, using genetics, biochemistry, and bioinformatics, we show that its essentiality derives from its role as a transcriptional repressor of the ydaS and ydaT genes, whose products are toxic to the cell. Unlike type II toxin-antitoxin systems in which transcriptional regulation involves complexes of the toxin and antitoxin, repression by RacR is sufficient to keep ydaS transcriptionally silent.

Constitutive Expression of a Transcription Termination Factor by a Repressed Prophage: Promoters for Transcribing the Phage HK022 nun Gene

PubMed Central

King, Rodney A.; Madsen, Peter L.; Weisberg, Robert A.

2000-01-01

Lysogens of phage HK022 are resistant to infection by phage λ. Lambda resistance is caused by the action of the HK022 Nun protein, which prematurely terminates early λ transcripts. We report here that transcription of the nun gene initiates at a constitutive prophage promoter, PNun, located just upstream of the protein coding sequence. The 5′ end of the transcript was determined by primer extension analysis of RNA isolated from HK022 lysogens or RNA made in vitro by transcribing a template containing the promoter with purified Escherichia coli RNA polymerase. Inactivation of PNun by mutation greatly reduced Nun activity and Nun antigen in an HK022 lysogen. However, a low level of residual activity was detected, suggesting that a secondary promoter also contributes to nun expression. We found one possible secondary promoter, PNun′, just upstream of PNun. Neither promoter is likely to increase the expression of other phage genes in a lysogen because their transcripts should be terminated downstream of nun. We estimate that HK022 lysogens in stationary phase contain several hundred molecules of Nun per cell and that cells in exponential phase probably contain fewer. PMID:10629193

Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

PubMed Central

Euler, Chad W.; Juncosa, Barbara; Ryan, Patricia A.; Deutsch, Douglas R.; McShan, W. Michael; Fischetti, Vincent A.

2016-01-01

Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy.

PubMed

Gai, Cláudia Santos; Lacava, Paulo Teixeira; Maccheroni, Walter; Glienke, Chirlei; Araújo, Welington Luiz; Miller, Thomas Albert; Azevedo, João Lúcio

2009-10-01

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of São Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were re-isolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms. Copyright 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

PubMed Central

Brüssow, Harald; Canchaya, Carlos; Hardt, Wolf-Dietrich

2004-01-01

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework. PMID:15353570

A force sensor using nanowire arrays to understand biofilm formation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sahoo, Prasana K.; Cavalli, Alessandro; Pelegati, Vitor B.; Murillo, Duber M.; Souza, Alessandra A.; Cesar, Carlos L.; Bakkers, Erik P. A. M.; Cotta, Monica A.

2016-03-01

Understanding the cellular signaling and function at the nano-bio interface can pave the way towards developing next-generation smart diagnostic tools. From this perspective, limited reports detail so far the cellular and subcellular forces exerted by bacterial cells during the interaction with abiotic materials. Nanowire arrays with high aspect ratio have been used to detect such small forces. In this regard, live force measurements were performed ex-vivo during the interaction of Xylella fastidiosa bacterial cells with InP nanowire arrays. The influence of nanowire array topography and surface chemistry on the response and motion of bacterial cells was studied in detail. The nanowire arrays were also functionalized with different cell adhesive promoters, such as amines and XadA1, an afimbrial protein of X.fastidiosa. By employing the well-defined InP nanowire arrays platform, and single cell confocal imaging system, we were able to trace the bacterial growth pattern, and show that their initial attachment locations are strongly influenced by the surface chemistry and nanoscale surface topography. In addition, we measure the cellular forces down to few nanonewton range using these nanowire arrays. In case of nanowire functionalized with XadA1, the force exerted by vertically and horizontally attached single bacteria on the nanowire is in average 14% and 26% higher than for the pristine array, respectively. These results provide an excellent basis for live-cell force measurements as well as unravel the range of forces involved during the early stages of bacterial adhesion and biofilm formation.

A novel chimeric prophage vB_LdeS-phiJB from commercial Lactobacillus delbrueckii subsp. bulgaricus.

PubMed

Guo, Tingting; Zhang, Chenchen; Xin, Yongping; Xin, Min; Kong, Jian

2016-05-01

Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated from a Chinese yoghurt sample. It has an isometric head and a non-contractile tail with 36,969 bp linear double-stranded DNA genome, which is classified into the group a of Lb. delbrueckii phages. The genome of phiJB is highly modular with functionally related genes clustered together. Unexpectedly, there is no similarity of its DNA replication module to any phages that have been reported, while it consists of open-reading frames homologous to the proteins of Lactobacillus strains. Comparative genomic analysis indicated that its late gene clusters, integration/lysogeny modules and DNA replication module derived from different evolutionary ancestors and integrated into a chimera. Our results revealed a novel chimeric phage of commercial Lb. delbrueckii and will broaden the knowledge of phage diversity in the dairy industry.

Egg maturation by the glassy-winged sharpshooter (Hemiptera: Cicadellidae); a vector of Xylella fastidiosa

USDA-ARS?s Scientific Manuscript database

Rates of spread of insect-transmitted plant pathogens are a function of vector abundance. Despite this, factors affecting population growth rates of insects that transmit plant pathogens have received limited attention. The glassy-winged sharpshooter (Homalodisca vitripennis) feeds on xylem-sap and ...

A genetic linkage map of grape, utilizing Vitis rupestris and Vitis arizonica.

PubMed

Doucleff, M; Jin, Y; Gao, F; Riaz, S; Krivanek, A F; Walker, M A

2004-10-01

A genetic linkage map of grape was constructed, utilizing 116 progeny derived from a cross of two Vitis rupestris x V. arizonica interspecific hybrids, using the pseudo-testcross strategy. A total of 475 DNA markers-410 amplified fragment length polymorphism, 24 inter-simple sequence repeat, 32 random amplified polymorphic DNA, and nine simple sequence repeat markers-were used to construct the parental maps. Markers segregating 1:1 were used to construct parental framework maps with confidence levels >90% with the Plant Genome Research Initiative mapping program. In the maternal (D8909-15) map, 105 framework markers and 55 accessory markers were ordered in 17 linkage groups (756 cM). The paternal (F8909-17) map had 111 framework markers and 33 accessory markers ordered in 19 linkage groups (1,082 cM). One hundred eighty-one markers segregating 3:1 were used to connect the two parental maps' parents. This moderately dense map will be useful for the initial mapping of genes and/or QTL for resistance to the dagger nematode, Xiphinema index, and Xylella fastidiosa, the bacterial causal agent of Pierce's disease.