Mutation of <i>R</i>. <i>solanacearum</i> strain UW551 <i>lecM</i>, <i>aidA</i>, or <i>aidC</i> but not <i>solI</i> differentially reduced bacterial virulence at 20°C.

<p>Virulence was measured on wilt-susceptible tomato plants at 20°C and 28°C via soil soak inoculation (A-E) or at 20°C via cut petiole inoculation (F). Each point represents the mean of three biological replicates, each containing 16 plants per strain per temperature. The area under disease progress curve (AUDPC) was measured for each strain in A-C and each mutant’s AUDPC relative to wild type is shown (E). Asterisks indicate that virulence of wild type and mutant strains were significantly different (* <i>P</i>< 0.05, ** <i>P</i>< 0.01, ANOVA). Each mutant was also significantly reduced in virulence (<i>P</i>< 0.01, repeated measures ANOVA) compared to the wild type following cut petiole inoculation (F).</p

Comparative Transcriptome Analysis Reveals Cool Virulence Factors of <i>Ralstonia solanacearum</i> Race 3 Biovar 2

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

UW551ΔlecM and UW551ΔaidC had reduced survival in potato tubers at 4°C.

<p>Potato tubers were injected with different <i>R</i>. <i>solanacearum</i> strains, and bacterial cell numbers were counted by grinding and dilution plating tubers at different times after inoculation. The experiment was repeated three times, with three tubers per strain per time point. At 6, 9 and 12 weeks after inoculation, the population sizes of UW551ΔlecM and UW551ΔaidC in tubers were significantly lower than those of the wild type parent strain (<i>P</i><0.05, ANOVA).</p

Expression of lectin genes in <i>R</i>. <i>solanacearum</i> strains GMI1000 and UW551 under various conditions.

<p>^aValues shown are scaled log2 signal intensities from strain-specific whole genome microarrays hybridized to labeled cDNA extracted from <i>R</i>. <i>solanacearum</i> cells grown in rich culture medium (CPG) or <i>in planta</i> (IP). These absolute expression values were used to allow comparisons across strains.</p><p>^b<i>np</i>: gene not present in this strain.</p><p>Expression of lectin genes in <i>R</i>. <i>solanacearum</i> strains GMI1000 and UW551 under various conditions.</p

A cluster of genes adjacent to those encoding the SolI/R quorum sensing system were up-regulated in <i>R</i>. <i>solanacearum</i> strain UW551 at 20°C, in culture.

<p>Some were also upregulated <i>in planta</i>. Arrows represent open reading frames. The numbers above the arrows indicate the expression fold-change for each gene at 20°C compared to 28°C, in culture/<i>in planta</i>, determined by whole-genome microarray analysis as described in the text. The arrangement and expression levels of the corresponding genes in strain GMI1000 are also shown.</p

Transcriptome Changes Associated with Anaerobic Growth in <i>Yersinia intermedia</i> (ATCC29909)

<div><p>Background</p><p>The yersiniae (Enterobacteriaceae) occupy a variety of niches, including some in human and flea hosts. Metabolic adaptations of the yersiniae, which contribute to their success in these specialized environments, remain largely unknown. We report results of an investigation of the transcriptome under aerobic and anaerobic conditions for <i>Y. intermedia</i>, a non-pathogenic member of the genus that has been used as a research surrogate for <i>Y. pestis</i>. <i>Y. intermedia</i> shares characteristics of pathogenic yersiniae, but is not known to cause disease in humans. Oxygen restriction is an important environmental stimulus experienced by many bacteria during their life-cycles and greatly influences their survival in specific environments. How oxygen availability affects physiology in the yersiniae is of importance in their life cycles but has not been extensively characterized.</p> <p>Methodology/Principal Findings</p><p>Tiled oligonucleotide arrays based on a draft genome sequence of <i>Y. intermedia</i> were used in transcript profiling experiments to identify genes that change expression in response to oxygen availability during growth in minimal media with glucose. The expression of more than 400 genes, constituting about 10% of the genome, was significantly altered due to oxygen-limitation in early log phase under these conditions. Broad functional categorization indicated that, in addition to genes involved in central metabolism, genes involved in adaptation to stress and genes likely involved with host interactions were affected by oxygen-availability. Notable among these, were genes encoding functions for motility, chemotaxis and biosynthesis of cobalamin, which were up-regulated and those for iron/heme utilization, methionine metabolism and urease, which were down-regulated.</p> <p>Conclusions/Significance</p><p>This is the first transcriptome analysis of a non-pathogenic <i>Yersinia</i><i>spp.</i> and one of few elucidating the global response to oxygen limitation for any of the yersiniae. Thus this study lays the foundation for further experimental characterization of oxygen-responsive genes and pathways in this ecologically diverse genus.</p> </div

Functional categories of anaerobically up-regulated (yellow) and anaerobically down-regulated (blue) genes.

<p>Genes from Table S1 were broadly categorized according to their biological function. Each bar represents the actual number of genes.</p

Graphical representation of 392 differentially expressed genes in Y. intermedia which have homologs in at least one of the pathogenic yersiniae.

<p>Phylogenprofiler (Integrated Microbial Genomes) was used with analytical settings as described in methods to obtain homologs of <i>Y</i>. <i>intermedia</i> in other yersiniae. A Venn diagram was built to display the number of differentially expressed genes in <i>Y</i>. <i>intermedia</i> that have homologs in <i>Y</i>. <i>enterocolitica</i> (<i>Ye8081</i>, blue circle, 376 genes), <i>Y</i>. <i>pseudotuberculosis</i> (<i>Ytb31758</i>, purple circle, 319 genes) and <i>Y</i>. <i>pestis</i> (<i>YpCO92</i>, red circle, 299 genes).</p

Volcano Plot of fold change versus significance.

<p>Gene expression data for <i>Y</i>. <i>intermedia</i> grown under aerobic and anaerobic conditions was used to derive log₂ ratios (X-axis) which are plotted against posterior probability of differential expression for each of the genes derived using EBarrays (Y-axis) to generate a volcano plot to visualize differential expression. Significant and insignificant genes are represented by black and grey diamonds, respectively. Red diamonds represent orthologs of genes identified as constituting the core anaerobic transcriptome of three enterobacterial members grown in the presence of glucose. A set of 20 genes were identified as likely to constitute the minimal core anaerobic transcriptome of the Enterobacteriaceae in the presence of glucose as the carbon source [22]. These 20 genes shared a 1-1-1 orthologous relationship between three members of the Enterobacteriaceae, namely <i>E</i>. <i>coli</i> K-12-MG1655, <i>Dickeya </i><i>dadantii</i> 3937 and <i>Pectobacterium </i><i>atrosepticum</i> SCRI1043 and for all of the 20 genes the pattern of expression was similar, the magnitude of change was greater than 3-fold and the genetic architecture was highly conserved. While the exact functions of most of these genes are established in the model organism <i>E</i>. <i>coli</i> that of few others still remain elusive. Of these 20 genes, 18 were differentially expressed and showed similar pattern of expression in <i>Y</i>. <i>intermedia</i> in this study. These are <i>frdABCD</i> (fumarate reductase), <i>focA, yfiD</i>, (pyruvate formate lyase), <i>adhE</i> (aldehyde dehydrogenases), <i>ynfK</i> (dethiobiotin synthetase)<i>, hypC</i> (hydrogenase components), <i>nrdD</i> (anaerobic ribonucleotide reductase), <i>dcuB</i> (dicarboxylate transporter), <i>yhbUV</i> (collagenase-like proteins), <i>pepT</i> (peptidase), <i>ycbJ</i> (uncharacterized protein), <i>exbB</i> (the membrane-spanning protein of the TonB-exbBD complex), <i>yceJ</i> (cytochrome), <i>yceI</i> (uncharacterized protein). Except for five genes (yfiD, yhbV,ycbJ, yceJ, yceI), all of the remaining 13 genes showed fold changes greater than 3 (our stringent criteria established in a previous study) in <i>Y</i>. <i>intermedia</i>. The only gene which is present in the core but missing in the differentially expressed set in <i>Y</i>. <i>intermedia</i> is <i>nrdG</i> (anaerobically functioning ribonucleotide reductase).</p