A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions.

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

Interplay between the RNA Chaperone Hfq, Small RNAs and Transcriptional Regulator OmpR Modulates Iron Homeostasis in the Enteropathogen Yersinia enterocolitica.

Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the fur, fecA and fepA genes, encoding the ferric uptake repressor and two transporters of ferric siderophores, respectively. This study was undertaken to determine the significance of the RNA chaperone Hfq and the small RNAs OmrA and RyhB1 in the post-transcriptional control of the expression of these OmpR targets. We show that Hfq silences fur, fecA and fepA expression post-transcriptionally and negatively affects the production of FLAG-tagged Fur, FecA and FepA proteins. In addition, we found that the fur gene is under the negative control of the sRNA RyhB1, while fecA and fepA are negatively regulated by the sRNA OmrA. Finally, our data revealed that the role of OmrA results from a complex interplay of transcriptional and post-transcriptional effects in the feedback circuit between the regulator OmpR and the sRNA OmrA. Thus, the expression of fur, fecA and fepA is subject to complex transcriptional and post-transcriptional regulation in order to maintain iron homeostasis in Y. enterocolitica.

Evolution of longitudinal division in multicellular bacteria of the Neisseriaceae family.

Rod-shaped bacteria typically elongate and divide by transverse fission. However, several bacterial species can form rod-shaped cells that divide longitudinally. Here, we study the evolution of cell shape and division mode within the family Neisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the genera Alysiella, Simonsiella and Conchiformibius, which can be found in the oral cavity of mammals, are multicellular and divide longitudinally. We use comparative genomics and ultrastructural microscopy to infer that longitudinal division within Neisseriaceae evolved from a rod-shaped ancestor. In multicellular longitudinally-dividing species, neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan. In these bacteria, peptidoglycan insertion does not appear concentric, i.e. from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, we identify genes and alleles associated with multicellularity and longitudinal division, including the acquisition of amidase-encoding gene amiC2, and amino acid changes in proteins including MreB and FtsA. Introduction of amiC2 and allelic substitution of mreB in a rod-shaped species that divides by transverse fission results in shorter cells with longer septa. Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria, and suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability.

The Regulatory Circuit Underlying Downregulation of a Type III Secretion System in Yersinia enterocolitica by Transcription Factor OmpR.

In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of Y. enterocolitica. Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes virF and yscM1, it highly repressed the expression of yopD. OmpR was found to bind to the promoter of the lcrGVsycD-yopBD operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of flhDC, the master regulator of the flagellar-associated T3SS.

Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis.

Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.

Urease Expression in Pathogenic Yersinia enterocolitica Strains of Bio-Serotypes 2/O:9 and 1B/O:8 Is Differentially Regulated by the OmpR Regulator.

Yersinia enterocolitica exhibits a dual lifestyle, existing as both a saprophyte and a pathogen colonizing different niches within a host organism. OmpR has been recognized as a regulator that controls the expression of genes involved in many different cellular processes and the virulence of pathogenic bacteria. Here, we have examined the influence of OmpR and varying temperature (26°C vs. 37°C) on the cytoplasmic proteome of Y. enterocolitica Ye9N (bio-serotype 2/O:9, low pathogenicity). Differential label-free quantitative proteomic analysis indicated that OmpR affects the cellular abundance of a number of proteins including subunits of urease, an enzyme that plays a significant role in acid tolerance and the pathogenicity of Y. enterocolitica. The impact of OmpR on the expression of urease under different growth conditions was studied in more detail by comparing urease activity and the transcription of ure genes in Y. enterocolitica strains Ye9N and Ye8N (highly pathogenic bio-serotype 1B/O:8). Urease expression was higher in strain Ye9N than in Ye8N and in cells grown at 26°C compared to 37°C. However, low pH, high osmolarity and the presence of urea did not have a clear effect on urease expression in either strain. Further analysis showed that OmpR participates in the positive regulation of three transcriptional units encoding the multi-subunit urease (ureABC, ureEF, and ureGD) in strain Ye9N, but this was not the case in strain Ye8N. Binding of OmpR to the ureABC and ureEF promoter regions was confirmed using an electrophoretic mobility shift assay, suggesting that this factor plays a direct role in regulating the transcription of these operons. In addition, we determined that OmpR modulates the expression of a ureR-like gene encoding a putative regulator of the ure gene cluster, but in the opposite manner, i.e., positively in Ye9N and negatively in Ye8N. These findings provide some novel insights into the function of OmpR in adaptation strategies of Y. enterocolitica.

OmpR-Mediated Transcriptional Regulation and Function of Two Heme Receptor Proteins of Yersinia enterocolitica Bio-Serotype 2/O:9.

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem-1 and weaker phem-2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the -35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem-1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5'-untranslated region (5'-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.

The Role of OmpR in the Expression of Genes of the KdgR Regulon Involved in the Uptake and Depolymerization of Oligogalacturonides in Yersinia enterocolitica.

Oligogalacturonide (OGA)-specific porins of the KdgM family have previously been identified and characterized in enterobacterial plant pathogens. We found that deletion of the gene encoding response regulator OmpR causes the porin KdgM2 to become one of the most abundant proteins in the outer membrane of the human enteropathogen Yersinia enterocolitica. Reporter gene fusion and real-time PCR analysis confirmed that the expression of kdgM2 is repressed by OmpR. We also found that kdgM2 expression is subject to negative regulation by KdgR, a specific repressor of genes involved in the uptake and metabolism of pectin derivatives in plant pathogens. The additive effect of kdgR and ompR mutations suggested that KdgR and OmpR regulate kdgM2 expression independently. We confirmed that kdgM2 occurs in an operon with the pelP gene, encoding the periplasmic pectate lyase PelP. A pectinolytic assay showed strong upregulation of PelP production/activity in a Y. enterocolitica strain lacking OmpR and KdgR, which corroborates the repression exerted by these regulators on kdgM2. In addition, our data showed that OmpR is responsible for up regulation of the kdgM1 gene encoding the second specific oligogalacturonide porin KdgM1. This indicates the involvement of OmpR in the reciprocal regulation of both KdgM1 and KdgM2. Moreover, we demonstrated the negative impact of OmpR on kdgR transcription, which might positively affect the expression of genes of the KdgR regulon. Binding of OmpR to the promoter regions of the kdgM2-pelP-sghX operon, and kdgM1 and kdgR genes was confirmed using the electrophoretic mobility shift assay, suggesting that OmpR can directly regulate their transcription. We also found that the overexpression of porin KdgM2 increases outer membrane permeability. Thus, OmpR-mediated regulation of the KdgM porins may contribute to the fitness of Y. enterocolitica in particular local environments.

Impact of OmpR on the membrane proteome of Yersinia enterocolitica in different environments: repression of major adhesin YadA and heme receptor HemR.

Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodelling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host.

Diversity and Global Distribution of IncL/M Plasmids Enabling Horizontal Dissemination of ß-Lactam Resistance Genes among the Enterobacteriaceae.

Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encoding ß-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain of Escherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encoding ß-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.

Expression of the AcrAB Components of the AcrAB-TolC Multidrug Efflux Pump of Yersinia enterocolitica Is Subject to Dual Regulation by OmpR.

OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in ß-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

Sequence determination and analysis of three plasmids of Pseudomonas sp. GLE121, a psychrophile isolated from surface ice of Ecology Glacier (Antarctica).

Pseudomonas sp. GLE121 (a psychrophilic Antarctic strain) carries three plasmids: pGLE121P1 (6899 bp), pGLE121P2 (8330 bp) and pGLE121P3 (39,583 bp). Plasmids pGLE121P1 and pGLE121P2 show significant sequence similarity to members of the IncP-9 and IncP-7 incompatibility groups, respectively, while the largest replicon, pGLE121P3, is highly related to plasmid pNCPPB880-40 of Pseudomonas syringae pathovar tomato NCPPB880. All three plasmids have a narrow host range, limited to members of the genus Pseudomonas. Plasmid pGLE121P3 encodes a conjugal transfer system, while pGLE121P1 carries only a putative MOB module, conserved in many mobilizable plasmids. Plasmid pGLE121P3 contains an additional load of genetic information, including a pair of genes with homology to the rulAB operon, responsible for ultraviolet radiation (UVR) tolerance. Given the increasing UV exposure in Antarctic regions, the expression of these genes is likely to be an important adaptive response.