Robust Heat Shock Response in Chlamydia Lacking a Typical Heat Shock Sigma Factor

Cells reprogram their transcriptome in response to stress, such as heat shock. In free-living bacteria, the transcriptomic reprogramming is mediated by increased DNA-binding activity of heat shock sigma factors and activation of genes normally repressed by heat-induced transcription factors. In this study, we performed transcriptomic analyses to investigate heat shock response in the obligate intracellular bacterium Chlamydia trachomatis, whose genome encodes only three sigma factors and a single heat-induced transcription factor. Nearly one-third of C. trachomatis genes showed statistically significant (≥1.5-fold) expression changes 30 min after shifting from 37 to 45°C. Notably, chromosomal genes encoding chaperones, energy metabolism enzymes, type III secretion proteins, as well as most plasmid-encoded genes, were differentially upregulated. In contrast, genes with functions in protein synthesis were disproportionately downregulated. These findings suggest that facilitating protein folding, increasing energy production, manipulating host activities, upregulating plasmid-encoded gene expression, and decreasing general protein synthesis helps facilitate C. trachomatis survival under stress. In addition to relieving negative regulation by the heat-inducible transcriptional repressor HrcA, heat shock upregulated the chlamydial primary sigma factor σ66 and an alternative sigma factor σ28. Interestingly, we show for the first time that heat shock downregulates the other alternative sigma factor σ54 in a bacterium. Downregulation of σ54 was accompanied by increased expression of the σ54 RNA polymerase activator AtoC, thus suggesting a unique regulatory mechanism for reestablishing normal expression of select σ54 target genes. Taken together, our findings reveal that C. trachomatis utilizes multiple novel survival strategies to cope with environmental stress and even to replicate. Future strategies that can specifically target and disrupt Chlamydia’s heat shock response will likely be of therapeutic value.

In planta transcriptomics reveals conflicts between pattern-triggered immunity and the AlgU sigma factor regulon

In previous work, we determined the transcriptomic impacts of flg22 pre-induced Pattern Triggered Immunity (PTI) in Arabidopsis thaliana on the pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). During PTI exposure we observed expression patterns in Pto reminiscent of those previously observed in a Pto algU mutant. AlgU is a conserved extracytoplasmic function sigma factor which has been observed to regulate over 950 genes in Pto in vitro. We sought to identify the AlgU regulon in planta.and which PTI-regulated genes overlapped with AlgU-regulated genes. In this study, we analyzed transcriptomic data from RNA-sequencing to identify the AlgU in planta regulon and its relationship with PTI. Our results showed that approximately 224 genes are induced by AlgU, while another 154 genes are downregulated by AlgU in Arabidopsis during early infection. Both stress response and virulence-associated genes were induced by AlgU, while the flagellar motility genes are downregulated by AlgU. Under the pre-induced PTI condition, more than half of these AlgU-regulated genes have lost induction/suppression in contrast to naive plants, and almost all function groups regulated by AlgU were affected by PTI.

The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella

Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.

Identification of genome-wide distribution of cyanobacterial group 2 sigma factor SigE accountable for its regulon

Sigma factors are the subunits of bacterial RNA polymerase that govern the expression of genes by recognizing the promoter sequence. Cyanobacteria, which are oxygenic phototrophic eubacteria, have multiple alternative sigma factors that respond to various environmental stresses. The subgroup highly homologous to the primary sigma factor (SigA) is called the group 2 sigma factor. The model cyanobacterium, Synechocystis sp. PCC 6803, has four group 2 sigma factors (SigB-E) conserved within the phylum Cyanobacteria. Among the group 2 sigma factors in Synechocystis sp. PCC 6803, SigE is unique because it alters metabolism by inducing the expression of genes related to sugar catabolism and nitrogen metabolism. However, the features of promoter sequence of the SigE regulon remains elusive. Here, we identified the direct targets of SigA and SigE by chromatin immunoprecipitation sequencing (ChIP-seq). We then showed that the binding sites of SigE and SigA overlapped substantially, but SigE exclusively localized to SigE-dependent promoters. We also found consensus sequences from SigE-dependent promoters and confirmed their importance. ChIP-seq analysis showed both the redundancy and specificity of SigE compared with SigA, integrating information obtained from a previously adopted genetic approach and in vitro assays. The features of SigE elucidated in our study indicate its similarity with group 2 sigma factors of other bacteria, even though they are evolutionally irrelevant. Our approach is also applicable to other organisms and organelles, such as plant plastids, which have multiple group 2 sigma factors.

Identification of transposon-inserted mutations including rnpB::Tn that abolished glucose induction of sigX encoding extracytoplasmic function-sigma factor in Bacillus subtilis

We investigated the regulators of the glucose induction (GI) of the ECF-sigma genes sigX/M. During further screening of transposon-inserted mutants, we identified several regulators including an RNA component of RNase P (rnpB), which is required for tRNA maturation. A depletion of rnpB is known to trigger the stringent response. We showed evidence that the stringent response inhibited GI of sigX/M.

The primary transcriptome of hormogonia from a filamentous cyanobacterium defined by cappable-seq

Hormogonia are motile filaments produced by many filamentous cyanobacteria that function in dispersal, phototaxis and the establishment of nitrogen-fixing symbioses. The gene regulatory network promoting hormogonium development is initiated by the hybrid histidine kinase HrmK, which in turn activates a sigma factor cascade consisting of SigJ, SigC and SigF. In this study, cappable-seq was employed to define the primary transcriptome of developing hormogonia in the model filamentous cyanobacterium Nostoc punctiforme ATCC 29133 in both the wild-type, and sigJ, sigC and sigF mutant strains 6 h post-hormogonium induction. A total of 1544 transcriptional start sites (TSSs) were identified that are associated with protein-coding genes and are expressed at levels likely to lead to biologically relevant transcripts in developing hormogonia. TSS expression among the sigma-factor deletion strains was highly consistent with previously reported gene expression levels from RNAseq experiments, and support the current working model for the role of these genes in hormogonium development. Analysis of SigJ-dependent TSSs corroborated the presence of the previously identified J-Box in the −10 region of SigJ-dependent promoters. Additionally, the data presented provides new insights on sequence conservation within the −10 regions of both SigC- and SigF-dependent promoters, and demonstrates that SigJ and SigC coordinate complex co-regulation not only of hormogonium-specific genes at different loci, but within an individual operon. As progress continues on defining the hormogonium gene regulatory network, this data set will serve as a valuable resource.