Search

Xylella fastidiosa is the causal agent of Pierce's disease (PD) in grapevines. It has type I and type IV pili, which are both virulence factors involved in the PD-associated processes of motility, aggregation, and biofilm formation. Many questions remain as to how the two pili are regulated. We previously identified a X. fastidiosa pil-chp chemosensory-like cluster as an operon composed of genes pilG-I-J-L-chpB-C. In this study, we deleted pilG (resulting in a ΔpilG-I strain) and pilJ and discovered that both mutants (ΔpilG-I and ΔpilJ) had reduced virulence after 24 weeks post-inoculation, whereas ΔchpB and ΔchpC did not. Both ΔpilG-I and ΔpilJ lost motility and were impaired in biofilm formation in rich artificial media and xylem sap. Gene expression was significantly downregulated for representative fimbrial adhesin and motility genes in ΔpilG-I, and to a lesser extent in ΔpilJ. Our data suggest that Pil, but not Chp, proteins are virulence factors, and pilG-I-J are involved in transcriptional regulation of type I and IV pili virulence genes and therefore motility and biofilm formation. To our knowledge, this is the first report of a chemotaxis-like operon involved in the regulation of key structural genes of both type I and type IV pili. [ABSTRACT FROM AUTHOR]

In Brazil mercury is used indiscriminately at prospecting sites for gold extraction, with a consequent enormous discharge of this metal into the aquatic ecosystem. Mercury concentration was investigated in 1995, 1996 and 1997 in the sediments of ten sites along the Carmo stream, MG, Brazil, almost all of them located in prospecting areas. Analysis of mercury in the sediments of all sampling sites showed that the levels were above the tolerable limit (0.1mu g g-1), except for sites P4 (1997), P6 (1995) and P9 (1995 and 1997). The results showed wide mercury contamination in an environment not limited tothe active or inactive prospecting areas, but also including sites outside these areas but downstream from them. There was a high incidence of bacteria resistant to mercury in the aquatic communities of thesites under study, ranging from 27.3 to 77.1%, except for P1 (an ecological station upstream from the sites under study) in which all bacteria isolated from water were sensitive. Furthermore, the fall in mercury concentration in the sediment at site P5 was not as marked as at other sites, with a frequency of resistant bacteria of only 27.3%, possibly indicating a slower detoxification. The statistical analysis(Pearson's correlation = ? 0,527) showed that the hypothesis about negative correlation between the incidence of bacterial resistance andthe total mercury concentration in this environment is supported. Hence, all these data denote a moderate association between the distribution of resistant bacteria and the presence of mercury compounds. [ABSTRACT FROM AUTHOR]

The aim of this study was to estimate genetic parameters for BW in meat quail at different ages. A total of 24,382 weight records from 3,652 quail, born between 2009 and 2011, were evaluated. Weekly BW was measured from hatch until 42 d of age. The genetic parameters were estimated by the restricted maximum likelihood method using a multivariate animal model. Heritability of BW ranged from 0.03 to 0.23. Genetic correlations were mainly high and positive. Selection for BW at 28 d of age yielded good indirect genetic progress in BW at 42 d of age. [ABSTRACT FROM AUTHOR]

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.)

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

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.

This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.

The characterization of Tn5 transposon insertional mutants of Agrobacterium vitis strain F2/5 revealed a gene encoding a predicted LysR-type transcriptional regulator, lhnR (for 'LysR-type regulator associated with HR and necrosis'), and an immediate upstream operon consisting of three open reading frames (lhnABC) required for swarming motility, surfactant production and the induction of a hypersensitive response (HR) on tobacco and necrosis on grape. The operon lhnABC is unique to A. vitis among the sequenced members in Rhizobiaceae. Mutagenesis of lhnR and lhnABC by gene disruption and complementation of ΔlhnR and ΔlhnABC confirmed their roles in the expression of these phenotypes. Mutation of lhnR resulted in complete loss of HR, swarming motility, surfactant production and reduced necrosis, whereas mutation of lhnABC resulted in loss of swarming motility, delayed and reduced HR development and reduced surfactant production and necrosis. The data from promoter-green fluorescent protein (gfp) fusions showed that lhnR suppresses the expression of lhnABC and negatively autoregulates its own expression. It was also shown that lhnABC negatively affects its own expression and positively affects the transcription of lhnR. lhnR and lhnABC constitute a regulatory circuit that coordinates the transcription level of lhnR, resulting in the expression of swarming, surfactant, HR and necrosis phenotypes., (© 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)

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.

We characterized indigenous common bean rhizobia from five districts of the state of Minas Gerais, Brazil. The isolates were trapped by two common bean varieties, the Mineiro Precoce (Andean origin) and Ouro Negro (Mesoamerican origin). Analysis by BOX-PCR of selected isolates detected a high level of genetic diversity.

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.

Three races of Xanthomonas axonopodis pv. glycines were identified on pustule disease resistant and susceptible soybean cultivars based on virulence phenotype. For race 3, an avrBs3 homolog, avrXg1 was identified that conferred resistance expressed as a hypersensitive response on resistant cultivar Williams 82. Mutations in two predicted functional domains of avrXg1 resulted in gained virulence on Williams 82 and an increase in bacterial population number on susceptible cultivars. Expression of avrXg1 in race 1, that is predicted to confer a nonspecific HR, led to virulence on susceptible cultivars Spencer and PI 520733. Expression of avrXg1 in race 2, that is predicted of carrying avrBs3-like genes, resulted in gained virulence and fitness of pathogen on both resistant and susceptible cultivars. The results demonstrate multifunctions for avrXg1 dependent on pathogen and plant genetic backgrounds.

Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P. stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.

The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014 bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion sequence) elements and transposons. Additional 14 ORFs code for proteins with similarities to known proteins (4 ORFs) or for hypothetical proteins with unknown function (10 ORFs). The differences in G+C content of individual ORFs and gene clusters of pMccC7-H22 indicate a mosaic structure for the plasmid, resulting from recombination events. Real-time PCR quantification was applied to measure the copy number of pMccC7-H22. Escherichia coli H22 carries approximately 5 copies of pMccC7-H22 per chromosome and thus pMccC7-H22 belongs to the group of relatively low-copy-number plasmids. Following 360 generations, all bacterial colonies (out of 100 tested) synthesized microcin C7 indicating that pMccC7-H22 is stably maintained in E. coli H22. Screening of 105 E. coli strains isolated from human fecal samples revealed 2 (1.9%) strains that produced microcin C7.

A freshwater enterobacterial population (N = 111) was studied for antimicrobial and mercury resistance patterns, and for its possible association with biotic and abiotic factors in that environment. Conventional biochemical tests identified Klebsiella sp, Morganella sp, Serratia sp, Escherichia sp, Enterobacter sp, Edwarsiella sp, Proteus sp, Citrobacter sp, Providencia sp, and Kluyvera sp. There was no correlation between antimicrobial resistance patterns of isolates and bacterial genera, but resistance patterns varied among water samples and between seasons. Resistance to multiple antimicrobials was common (61%). The percentage of bacteria resistant to at least one antimicrobial differed between the rainy (100%) and dry seasons (89%). Resistance to beta-lactams and chloramphenicol was the most frequent and resistance to amikacin, gentamicin and kanamycin was less frequent. The main water variables examined (abiotic factors pH and temperature; biotic factor chlorophyll a concentration) did not influence antimicrobial resistance. Significant impact on freshwater enterobacteria, as evidenced by antimicrobial-multiple resistance and by the presence of bla(TEM) gene, may point to the fact that it has an important role in horizontal spread of resistance.