Most microbial communities consist of a genetically diverse assembly of different organisms, and the level of genetic diversity plays an important part in community properties and functions. However, biological diversity also arises at a lower level of biological organization, between genetically identical cells that reside in the same microenvironment. In this Review, I outline the molecular mechanisms responsible for phenotypic heterogeneity and discuss how phenotypic heterogeneity allows genotypes to persist in fluctuating environments. I also describe how it promotes interactions between phenotypic subpopulations in clonal groups, providing microbial groups with new functionality.

A functional perspective on phenotypic heterogeneity in microorganisms

To survive in rapidly changing environmental conditions, bacteria have evolved a diverse set of regulatory pathways that govern various adaptive responses. Recent research has reinforced the notion that bacteria use feedback-based circuitry to generate population heterogeneity in natural situations. Using artificial gene networks, it has been shown that a relatively simple 'wiring' of a bacterial genetic system can generate two or more stable subpopulations within an overall genetically homogeneous population. This review discusses the ubiquity of these processes throughout nature, as well as the presumed molecular mechanisms responsible for the heterogeneity observed in a selection of bacterial species.

/pdf/phenotypic-variation-in-bacteria-the-role-of-feedback-5mwplpgp6h.pdf

Phenotypic variation in bacteria: the role of feedback regulation

The increasing incidence of infectious diseases is driving renewed efforts to understand the interactions between bacterial pathogens and their hosts at a new level. This has prompted research to study the transcriptional response of bacteria during infection. The past decade has seen the development of a number of techniques for identifying in vivo-induced bacterial genes and for monitoring gene induction in complex environments. Genetic tools for visualization of gene expression in situ have also been devised. Fluorescent reporters have been developed for studying bacterial infection from both the host point of view and the bacterial point of view (44, 57, 60). The green fluorescent protein (GFP) from Aequoria victoria is an excellent tool for monitoring gene expression, because it is naturally fluorescent without any exogenous cofactor or substrate (9). Variants of GFP that have different spectral characteristics, folding properties, levels of chromophore brightness, or half-lives have broadened the spectrum of possible applications for GFP (4). Measurement of fluorescence by flow cytometry has revolutionized the use of fluorescent reporter genes in bacteria (27) and now allows observation of gene expression at the level of individual bacterial cells. Plasmid-borne gfp fusions have already been used to monitor gene expression in Salmonella strains (6, 61), as well as in other gram-negative or gram-positive bacteria, including Escherichia coli (42), Serratia liquefaciens (2), Erwinia herbicola (37), Staphylococcus aureus (45), Listeria monocytogenes (64), and Streptococcus gordonii (24). Such fusions have proven to be useful for identifying bacterial genes induced during infection of eukaryotic cells or animal hosts and have allowed some assessment of bacterial gene expression levels in such complex environments. However, the variation in plasmid copy number that can occur in individual bacterial cells makes plasmid-borne gene fusions unsuitable for measuring differences in gene expression between single bacteria (34, 35). Furthermore, plasmid-borne GFP fusions are useful only for reporting the induction of genes; any reduction in fluorescence may simply reflect plasmid loss, because it is not possible to guarantee that plasmids are maintained in every bacterial cell in plant or animal hosts. Indeed, high expression levels lead to accumulation of toxic levels of GFP in bacterial cells, which results in dramatic plasmid loss (63; I. Hautefort, J. M. Sidebotham, and J. C. D. Hinton, unpublished data). Hopkins et al. showed that a pBR322-derived plasmid-borne gfp fusion was unstable in wild-type Salmonella enterica serovar Typhimurium strain 14028 during infection of BALB/c mice, while it remained stable in a PhoPc mutant (29). More recently, it was reported that a plasmid-borne tac-gfp fusion was not stable in a wild-type Salmonella strain during murine infection (6). This study showed that the level of plasmid loss was related to promoter strength, meaning that the levels of expression of different promoters cannot be compared by using plasmid-borne gene fusions.

Our interest in determining the diversity of gene expression within bacterial populations in the host prompted us to develop an improved approach for construction of gfp transcriptional fusions that could be used reliably during infection. We reasoned that integration of single-copy gene fusions onto the bacterial chromosome was the best way to ensure genetic stability. Single-copy gfp gene fusions have been used to monitor the activity of the marRAB promoter in large populations of Salmonella serovar Typhimurium (46). This approach involves inactivation of the gene of interest, making it unsuitable for studying virulence genes during infection. Recently, the activity of the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible spac hybrid promoter (65) was successfully monitored by using a single copy of gfpmut2 as a reporter in Bacillus subtilis (41). We performed preliminary experiments involving expression of single-copy gfpmut1 fusions in Salmonella; and we discovered that the GFPmut1 variant, which is even brighter than GFPmut2 (11), was only fluorescent enough to report particularly high levels of promoter activity in Salmonella during infection (Hautefort et al., unpublished). We overcame this problem by using the GFP+ variant (51), which carries the GFPuv mutations F99S, M153T, and V163A (12) along with the EGFP mutations F64L and S65T (11), resulting in better folding of the protein coupled with enhanced brightness. In this study we describe the use of GFP+-based single-copy gene fusions to measure Salmonella serovar Typhimurium gene induction in vitro and during infection of mammalian cells.

Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells.

Genetic susceptibility, the mucosal immune system, and environmental factors such as the microbiota, stress, and diet, contribute to the pathogenesis of inflammatory bowel disease (IBD) (
15
) Involvement of the microbiota has been proposed early on, as microbiota manipulation by antibiotics or probiotics can treat or alleviate IBD symptoms in humans In experimental animal models, the gut luminal microbiota is required for the induction of chronic inflammation (
16
) Different theories about how the microbiota is involved in the pathogenesis of IBD have been proposed (1) Mutations that lead to a defective mucosal barrier function (eg, mucus layer, innate killing, antimicrobial peptides) involve excessive translocation of commensal bacteria and triggering of proinflammatory signalling cascades (2) Abnormal host immune regulation induces an overshooting immune response against intrinsic commensal bacteria (3) The presence of an unidentified pathogen leads to induction of the disease or (4), a dysbiotic microbiota, characterized by an imbalance between “beneficial” and “potentially harmful” commensal bacteria, acts as a trigger or driver of the disease The latter theory has been challenged by studies conducted in experimental rodent models: inflammatory disease conditions in the course of chronic colitis or enteropathogen infection can disrupt normal microbiota composition, induce dysbiosis, and favor overgrowth of pathogens and commensals with an increased virulence potential (
10
, 
14
, 
17
) Therefore, dysbiosis may not only be considered as a cause but also a consequence of gut inflammation

The Roles of Inflammation, Nutrient Availability and the Commensal Microbiota in Enteric Pathogen Infection.

The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins.

/pdf/a-novel-immunity-system-for-bacterial-nucleic-acid-degrading-2xtogwmvgj.pdf

A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems

In prokaryotes, only a few examples of differential gene expression in cell populations have been described. Colicin production in natural populations of Escherichia coli, while providing a competitive advantage in the natural habitat, also leads to lysis of the toxin-producing cell. Colicin K synthesis has been found to be induced due to an increase in ppGpp (I. Kuhar, J. P. van Putten, D. Žgur-Bertok, W. Gaastra, and B. J. Jordi, Mol. Microbiol. 41:207-216). Using two transcriptional fusions, cka-gfp and cki-gfp, we show that at the single-cell level, the colicin K activity gene cka is expressed in only 3% of the bacterial population upon induction by nutrient starvation. In contrast, the immunity gene cki is expressed in the large majority of the cells. Expression of the cka-gfp fusion in a lexA-defective strain and in a relA spoT mutant strain indicates that differential expression of cka is established primarily at the level of transcription.

A cka-gfp transcriptional fusion reveals that the colicin K activity gene is induced in only 3 percent of the population

Phenotypic diversity provides populations of prokaryotic and eukaryotic organisms with the flexibility required to adapt to and/or survive environmental perturbations. Consequently, there is much interest in unraveling the molecular mechanisms of heterogeneity. A classical example of heterogeneity in Escherichia coli is the subset (3%) of the population that expresses the colicin K activity gene (cka) upon nutrient starvation. Here, we report on the mechanism underlying this variable response. As colicin synthesis is regulated by the LexA protein, the central regulator of the SOS response, we focused on the role of LexA and the SOS system in the variable cka expression. Real-time RT-PCR showed that the SOS system, without exogenous DNA damage, induces moderate levels of cka expression. The use of cka-gfp fusions demonstrated that modification of the conserved LexA boxes in the cka promoter region affected LexA binding affinity and the percentage of cka-gfp expressing cells in the population. A lexA-gfp fusion showed that the lexA gene is highly expressed in a subset of bacteria. Furthermore, cka-gfp fusions cloned into higher copy plasmid vectors increased the percentage of cka-gfp positive bacteria. Together, these results indicate that the bistability in cka expression in the bacterial population is determined by (1) basal SOS activity, (2) stochastic factors and possibly (3) the interplay of LexA dimers at cka operator. Other LexA regulated processes could exhibit similar regulation.

/pdf/heterogeneity-in-expression-of-the-escherichia-coli-colicin-xgiiheuavs.pdf

Heterogeneity in expression of the Escherichia coli colicin K activity gene cka is controlled by the SOS system and stochastic factors.

Colicin K exhibited pronounced inhibitory activity against uropathogenic Escherichia coli (UPEC) strains. Low prevalence of colicin K production and a relatively high prevalence of ColE1-like plasmids were determined among 215 UPEC strains from Slovenia. Sequencing of the colicin K-encoding pColK-K235 revealed a mosaic structure and the presence of the insertion sequence IS2.

Prevalence of ColE1-Like Plasmids and Colicin K Production among Uropathogenic Escherichia coli Strains and Quantification of Inhibitory Activity of Colicin K

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A cka-gfp transcriptional fusion reveals that the colicin K activity gene is induced in only 3 percent of the population

Heterogeneity in expression of the Escherichia coli colicin K activity gene cka is controlled by the SOS system and stochastic factors.

Prevalence of ColE1-Like Plasmids and Colicin K Production among Uropathogenic Escherichia coli Strains and Quantification of Inhibitory Activity of Colicin K