Transcriptome changes associated with anaerobic growth in Yersinia intermedia (ATCC29909)

Abstract

Background: 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 Y. intermedia, a non-pathogenic member of the genus that has been used as a research surrogate for Y. pestis. Y. intermedia 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.

Methodology/principal findings: Tiled oligonucleotide arrays based on a draft genome sequence of Y. intermedia 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.

Conclusions/significance: This is the first transcriptome analysis of a non-pathogenic Yersinia spp. 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.

Figure 1. Volcano Plot of fold change versus significance.

Gene expression data for Y. intermedia 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 E. coli K-12-MG1655, Dickeya dadantii 3937 and Pectobacterium atrosepticum 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 E. coli that of few others still remain elusive. Of these 20 genes, 18 were differentially expressed and showed similar pattern of expression in Y. intermedia in this study. These are frdABCD (fumarate reductase), focA, yfiD, (pyruvate formate lyase), adhE (aldehyde dehydrogenases), ynfK (dethiobiotin synthetase), hypC (hydrogenase components), nrdD (anaerobic ribonucleotide reductase), dcuB (dicarboxylate transporter), yhbUV (collagenase-like proteins), pepT (peptidase), ycbJ (uncharacterized protein), exbB (the membrane-spanning protein of the TonB-exbBD complex), yceJ (cytochrome), yceI (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 Y. intermedia. The only gene which is present in the core but missing in the differentially expressed set in Y. intermedia is nrdG (anaerobically functioning ribonucleotide reductase).

Figure 2. Graphical representation of 392 differentially expressed genes in Y. intermedia which have homologs in at least one of the pathogenic yersiniae.

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

Figure 3. Functional categories of anaerobically up-regulated (yellow) and anaerobically down-regulated (blue) genes.

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