Regulation of Gene Expression of phiEco32-like Bacteriophage 7-11

Salmonella enterica serovar Newport bacteriophage 7-11 shares 41 homologous ORFs with Escherichia coli phage phiEco32 and both phages encode a protein similar to bacterial RNA polymerase promoter specificity  subunit. Here, we investigated the temporal pattern of 7-11 gene expression during the infection and compared it to the previously determined transcription strategy of phiEco32. Using primer extension and in vitro transcription assays we identified eight promoters recognized by host RNA polymerase holoenzyme containing 7-11  subunit SaPh711_gp47. These promoters are characterized by a bipartite consensus GTAAtg-(16)-aCTA and are located upstream of late phage genes. While dissimilar from single-element middle and late promoters of phiEco32 recognized by holoenzyme formed by the phi32_gp36  factor, the 7-11 late promoters are located at genome positions similar to those of phiEco32 middle and late promoters. Two early 7-11 promoters are recognized by RNA polymerase holoenzyme containing host primary σ70 factor. Unlike the case of phiEco32, no shut off of σ70-dependent transcription is observed during 7-11 infection and there are no middle promoters. These differences can be explained by the fact that phage 7-11 does not encode a homologue of phi32_gp79, an inhibitor of host and early phage transcription and an activator of transcription by the phi32_gp36-holoenzyme.

Regulatory small RNA, Qrr2 is expressed independently of sigma factor-54 and can function as the sole Qrr sRNA to control quorum sensing in Vibrio parahaemolyticus

Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi , LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus , the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ luxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a Δ rpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in Δ luxO showed all five qrr genes were repressed, while in Δ rpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species indicating it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ rpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR . Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2 . Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation. Importance The quorum sensing non-coding sRNAs are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in V. harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in V. cholerae the qrr genes are redundant in function. In V. parahaemolyticus , qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR.

Regulatory small RNA, Qrr2, is expressed independently of sigma factor-54 and functions autonomously in Vibrio parahaemolyticus to control quorum sensing

Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), required for capsule polysaccharide (CPS) and biofilm production, motility, and metabolism. We show that in a ΔluxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a ΔrpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in ΔluxO showed all five qrr genes were repressed, while in ΔrpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act autonomously to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) promoter in the qrr2 regulatory region. Mutagenesis of the sigma-70 -10 promoter site in the ΔrpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act autonomously to regulate opaR. Mutant and expression data also demonstrated that RpoN and the global regulator Fis act additively to repress qrr2. Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation.

Transcriptional Landscape of Staphylococcus aureus Kayvirus Bacteriophage vB_SauM-515A1

The Twort-like myoviruses (Kayvirus genus) of S. aureus are promising agents for bacteriophage therapy due to a broad host range and high killing activity against clinical isolates. This work improves the current understanding of the phage infection physiology by transcriptome analysis. The expression profiles of a typical member of the Kayvirus genus (vB_SauM-515A1) were obtained at three time-points post-infection using RNA sequencing. A total of 35 transcription units comprising 238 ORFs were established. The sequences for 58 early and 12 late promoters were identified in the phage genome. The early promoters represent the strong sigma-70 promoters consensus sequence and control the host-dependent expression of 26 transcription units (81% of genes). The late promoters exclusively controlled the expression of four transcription units, while the transcription of the other five units was directed by both types of promoters. The characteristic features of late promoters were long -10 box of TGTTATATTA consensus sequence and the absence of -35 boxes. The data obtained are also of general interest, demonstrating a strategy of the phage genome expression with a broad overlap of the early and late transcription phases without any middle transcription, which is unusual for the large phage genomes (>100 kbp).

Characterisation and Mutagenesis Study of An Alternative Sigma Factor Gene (hrpL) from Erwinia mallotivora Reveal Its Central Role in Papaya Dieback Disease

The alternative sigma (σ) factor E, RpoE or HrpL, has been reported to be involved in stress- and pathogenicity-related transcription initiation in Escherichia coli and many other Gram-negative bacteria, including Erwinia spp. and Pseudomonas spp. A previous study identified the hrpL/rpoE transcript as one of the significant differentially expressed genes (DEGs) during early E. mallotivora infection in papaya and those data serve as the basis of the current project. Here, the full coding DNA sequence (CDS) of hrpL from E. mallotivora (EmhrpL) was determined to be 549 bp long, and it encoded a 21.3 kDa HrpL protein that possessed two highly conserved sigma-70 (σ70) motifs—σR2 and σR4. Nucleotide sequence alignment revealed the hrpL from E. mallotivora shared high sequence similarity to rpoE/hrpL from E. tracheiphila (83%), E. pyrifoliae (81%), and E. tasmaniensis (80%). Phylogenetics analysis indicated hrpL from E. mallotivora to be monophyletic with rpoEs/hrpLs from Pantoea vagans, E. herbicola, and E. tracheiphila. Structural analysis postulated that the E. mallotivora’s alternative σ factor was non-transmembranic and was an extracytoplasmic function (ECF) protein—characteristics shared by other σ factors in different bacterial species. Notably, the protein–protein interaction (PPI) study through molecular docking suggested the σ factor could be possibly inhibited by an anti-σ. Finally, a knockout of hrpL in E. mallotivora (ΔEmhrpL) resulted in avirulence in four-month-old papaya plants. These findings have revealed that the hrpL is a necessary element in E. mallotivora pathogenicity and also predicted that the gene can be inhibited by an anti-σ.

Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory

ABSTRACTEctoine and hydroxyectoine are widely synthesized by members of theBacteriaand a few members of theArchaeaas potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacteriumPseudomonas stutzeriby transferring it intoEscherichia coli, an enterobacterium that does not produce ectoines naturally. Usingect-lacZreporter fusions, we found that the heterologousectpromoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the −10, −35, and spacer regions of the sigma-70-typeectpromoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in theectpromoter sequence that increase its resemblance to housekeeping sigma-70-type promoters ofE. coliafforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set ofectpromoter mutants, we engineered anE. colichassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions.IMPORTANCEEctoines are compatible solutes, organic osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited theectpromoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact thatE. colidoes not synthesize ectoines naturally, theectpromoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation ofecttranscription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116-bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of theectpromoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.

Strong spurious transcription likely a cause of DNA insert bias in typical metagenomic clone libraries

Background: Clone libraries provide researchers with a powerful resource with which to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, as well as allowed the mining of novel enzymes for specific functions of interest. These libraries are often constructed by cloning large-inserts (~30 kb) into a cosmid or fosmid vector. Recently, there have been reports of GC bias in fosmid metagenomic clone libraries, and it was speculated that the bias may be a result of fragmentation and loss of AT-rich sequences during the cloning process. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role in library bias. Results: To explore the possible mechanisms responsible for sequence bias in clone libraries, and in particular whether fragmentation is involved, we constructed a cosmid clone library from a human microbiome sample, and sequenced DNA from three different steps of the library construction process: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final constructed cosmid library, and we provide strong evidence that the sequence bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after the DNA is introduced into E. coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for consensus promoters and found that rpoD/sigma-70 promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the reference genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found that the bias appears to be more closely correlated with the number of rpoD/sigma-70 consensus sequences in the genome than with simple GC content. Conclusions: The GC bias of metagenomic clone libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter consensus sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/sigma-70 consensus-like in E. coli may lead to plasmid instability or loss of insert DNA. Our results suggest that despite widespread use of E. coli to propagate foreign DNA, the effects of in vivo transcriptional activity may be under-appreciated. Further work is required to tease apart the effects of transcription from those of gene product toxicity.