Andrew Free

Anti-silencing : overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

The H-NS nucleoid-associated DNA-binding protein is an important global repressor of transcription in Gram-negative bacteria. Recently, H-NS has been implicated in the process of xenogeneic silencing, where it represses the transcription of foreign genes acquired by horizontal transfer. This raises interesting questions about the integration of the horizontally acquired genes into the existing gene regulatory networks of the microbe. In particular, how do bacteria derepress silenced genes in order to benefit from their expression without compromising competitive fitness through doing so inappropriately? This article reviews current knowledge about the derepression of genes that are transcriptionally silenced by H-NS. It describes a variety of anti-silencing mechanisms involving (i) protein-independent processes that operate at the level of local DNA structure, (ii) DNA-binding proteins such as Ler, LeuO, RovA, SlyA, VirB, and proteins related to AraC, and (iii) modulatory mechanisms in which H-NS forms heteromeric protein-protein complexes with full-length or partial paralogues such as StpA, Sfh, Hha, YdgT, YmoA or H-NST. The picture that emerges is one of apparently ad hoc solutions to the problem of H-NS-mediated silencing, suggesting that microbes are capable of evolving anti-silencing methods based on the redeployment of existing regulatory proteins rather than employing dedicated, bespoke antagonists. There is also evidence that in a number of cases more sophisticated regulatory processes have been superimposed on these rather simple anti-silencing mechanisms, broadening the range of environmental signals to which H-NS-repressed genes respond.

Domain organization and oligomerization among H-NS-like nucleoid-associated proteins in bacteria

The bacterial nucleoid-associated proteins H-NS and StpA can form homomeric or heteromeric complexes, a parallel with protein HU. Thus, functional modulation of H-NS and StpA by one another and by other proteins with appropriate interaction domains is possible. This has implications for bacterial pathogenesis and adaptation to environmental stress.

Challenges in microbial ecology: building predictive understanding of community function and dynamics

The importance of microbial communities (MCs) cannot be overstated. MCs underpin the biogeochemical cycles of the earth’s soil, oceans and the atmosphere, and perform ecosystem functions that impact plants, animals and humans. Yet our ability to predict and manage the function of these highly complex, dynamically changing communities is limited. Building predictive models that link MC composition to function is a key emerging challenge in microbial ecology. Here, we argue that addressing this challenge requires close coordination of experimental data collection and method development with mathematical model building. We discuss specific examples where model–experiment integration has already resulted in important insights into MC function and structure. We also highlight key research questions that still demand better integration of experiments and models. We argue that such integration is needed to achieve significant progress in our understanding of MC dynamics and function, and we make specific practical suggestions as to how this could be achieved.

Novel Electrochemically Active Bacterium Phylogenetically Related to Arcobacter butzleri, Isolated from a Microbial Fuel Cell

ABSTRACT Exoelectrogenic bacteria are organisms that can transfer electrons to extracellular insoluble electron acceptors and have the potential to be used in devices such as microbial fuel cells (MFCs). Currently, exoelectrogens have been identified in the Alpha-, Beta-, Gamma- and Deltaproteobacteria, as well as in the Firmicutes and Acidobacteria. Here, we describe use of culture-independent methods to identify two members of the genus Arcobacter in the Epsilonproteobacteria that are selectively enriched in an acetate-fed MFC. One of these organisms, Arcobacter butzleri strain ED-1, associates with the electrode and rapidly generates a strong electronegative potential as a pure culture when it is supplied with acetate. A mixed-community MFC in which ∼90% of the population is comprised of the two Arcobacter species generates a maximal power density of 296 mW/liter. This demonstration of exoelectrogenesis by strain ED-1 is the first time that this property has been shown for members of this genus.

Direct and indirect transcriptional activation of virulence genes by an AraC‐like protein, PerA from enteropathogenic Escherichia coli

The plasmid‐encoded Per regulatory locus of enteropathogenic Escherichia coli (EPEC) is generally considered to consist of three genes, perA, perB and perC. PerA, a member of the AraC‐like family of transcriptional regulators, is known to be an activator of its own promoter (autoactivation) as well as of the plasmid‐located bfp operon encoding bundle‐forming pili, but its role in activation of the chromosomal locus of enterocyte effacement (LEE) pathogenicity island, which confers the property of intimate adherence on EPEC, requires clarification. Here, we show that PerA is also required for activation of the master regulatory LEE operon, LEE1, but that this activation is indirect, being achieved via autoactivation of the per promoter which ensures sufficient production of the PerC protein to activate LEE1. In contrast, PerA‐dependent activation of the per and bfp promoters is direct and does not require the other Per proteins, but is modulated by the nucleoid‐associated protein H‐NS. The closely related VirF regulator from Shigella flexneri cannot substitute for PerA to activate these promoters, despite being able to bind their upstream regions in vitro. PerA can bind the per and bfp promoter fragments to form multiple complexes, while VirF forms only a single complex. Site‐directed mutagenesis of the PerA protein suggests that, like VirF, it may use both of its carboxy‐terminal helix—turn–helix motifs for DNA interaction, and may also make direct contacts with RNA polymerase. In addition, we have isolated mutations in the poorly characterized amino‐terminal domain of PerA which affect its ability to activate gene expression.

The LEE1 promoters from both enteropathogenic and enterohemorrhagic Escherichia coli can be activated by PerC-like proteins from either organism

The PerC protein of enteropathogenic Escherichia coli (EPEC), encoded by the pEAF plasmid, is an activator of the locus of enterocyte effacement (LEE) pathogenicity island via the LEE1 promoter. It has been assumed that the related LEE-containing pathogen enterohemorrhagic E. coli (EHEC) lacks PerC-dependent activation due to utilization of an alternative LEE1 promoter and lack of a perC gene. However, we show here that EPEC PerC can activate both the EPEC and EHEC LEE1 promoters and that the major transcriptional start site is similarly located in both organisms. Moreover, a PerC-like protein family identified from EHEC genome analyses, PerC1 (also termed PchABC), can also activate both promoters in a manner similar to that of EPEC PerC. The perC1 genes are carried by lambdoid prophages, which exist in multiple copies in different EHEC strains, and have a variable flanking region which may affect their expression. Although individual perC1 copies appear to be poorly expressed, the total perC1 expression level from a strain encoding multiple copies approaches that of perC in EPEC and may therefore contribute significantly to LEE1 activation. Alignment of the protein sequences of these PerC homologues allows core regions of the PerC protein to be identified, and we show by site-directed mutagenesis that these core regions are important for function. However, purified PerC protein shows no in vitro binding affinity for the LEE1 promoter, suggesting that other core E. coli proteins may be involved in its mechanism of activation. Our data indicate that the nucleoid-associated protein IHF is one such protein.

A truncated H‐NS‐like protein from enteropathogenic Escherichia coli acts as an H‐NS antagonist

The H‐NS nucleoid‐associated protein of Escherichia coli is the founder member of a widespread family of gene regulatory proteins which have a bipartite structure, consisting of an N‐terminal coiled‐coil oligomerization domain and a C‐terminal DNA‐binding domain. Here we characterize a family of naturally occurring truncated H‐NS derivatives lacking the DNA‐binding domain, which we term the H‐NST family. H‐NST proteins are found in large genomic islands in pathogenic E. coli strains, which are absent from the corresponding positions in the E. coli K‐12 genome. Detailed analysis of the H‐NST proteins from enteropathogenic E. coli (EPEC) and uropathogenic E. coli (UPEC) shows that the EPEC protein (H‐NSTEPEC) has a potent anti‐H‐NS function at the classical H‐NS‐repressed operon proU. This correlates with the ability of H‐NSTEPEC to co‐purify with H‐NS in vitro, and can be abolished by a mutation of leucine 30 to proline which is predicted to prevent the N‐terminal region from forming a coiled‐coil structure. In contrast, despite being 90% identical to H‐NSTEPEC at the protein level, the UPEC homologue (H‐NSTUPEC) has only a weak anti‐H‐NS activity, correlating with a much‐reduced ability to interact with H‐NS during column chromatography. A single amino acid difference at residue 16 appears to account for these different properties. The hnsTEPEC gene is transcribed monocistronically and expressed throughout the exponential growth phase in DMEM medium. Our data suggest that a truncated derivative of H‐NS encoded by an ancestral mobile DNA element can interact with the endogenous H‐NS regulatory network of a bacterial pathogen.

Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein.

Truncated derivatives of the Escherichia coli nucleoid‐associated protein H‐NS that lack the DNA‐binding domain remain competent for silencing of the cryptic bgl operon in vivo. Previous studies have provided evidence for the involvement of either the homologous nucleoid protein StpA or the alternative sigma factor RpoS in this unusual silencing mechanism. Here, we rationalize this apparent discrepancy. We show that two hns alleles (hns‐205::Tn10 and hns60), which produce virtually identical amino‐terminal fragments of H‐NS, have very different requirements for StpA to mediate bgl silencing. The hns60 allele produces a high level of truncated H‐NS, which can overcome the absence of StpA, whereas the lower level expressed by hns‐205::Tn10 requires StpA for silencing. Reversing the relative levels of the two H‐NS fragments reverses their requirement for StpA to silence bgl transcription. This suggests that the amino‐terminal fragment of H‐NS can be targeted to DNA to mediate silencing by multiple protein–protein interactions. The high‐specificity interaction with StpA can function at low levels of truncated H‐NS, whereas an alternative mechanism, perhaps involving lower specificity interactions with another protein(s), is only functional when truncated H‐NS is abundant. These findings have important implications for the involvement of other proteins in H‐NS‐dependent transcriptional repression.

Community history affects the predictability of microbial ecosystem development

Microbial communities mediate crucial biogeochemical, biomedical and biotechnological processes, yet our understanding of their assembly, and our ability to control its outcome, remain poor. Existing evidence presents conflicting views on whether microbial ecosystem assembly is predictable, or inherently unpredictable. We address this issue using a well-controlled laboratory model system, in which source microbial communities colonize a pristine environment to form complex, nutrient-cycling ecosystems. When the source communities colonize a novel environment, final community composition and function (as measured by redox potential) are unpredictable, although a signature of the community’s previous history is maintained. However, when the source communities are pre-conditioned to their new habitat, community development is more reproducible. This situation contrasts with some studies of communities of macro-organisms, where strong selection under novel environmental conditions leads to reproducible community structure, whereas communities under weaker selection show more variability. Our results suggest that the microbial rare biosphere may have an important role in the predictability of microbial community development, and that pre-conditioning may help to reduce unpredictability in the design of microbial communities for biotechnological applications.

The microbiota and helminths: sharing the same niche in the human host

Human gastrointestinal bacteria often share their environment with parasitic worms, allowing physical and physiological interaction between the two groups. Such associations have the potential to affect host health as well as the bacterial and helminth populations. Although still in its early stages, research on the interaction between the microbiome and parasitic helminths in humans offers the potential to improve health by manipulating the microbiome. Previously, supplementation with various nutritional compounds has been found to increase the abundance of potentially beneficial gut commensal bacteria. Thus, nutritional microbiome manipulation to produce an environment which may decrease malnutrition associated with helminth infection and/or aid host recovery from disease is conceivable. This review discusses the influence of the gut microbiota and helminths on host nutrition and immunity and the subsequent effects on the human hosts overall health. It also discusses changes occurring in the microbiota upon helminth infections and the underlying mechanisms leading to these changes. There are still significant knowledge gaps which need to be filled before meaningful progress can be made in translating knowledge from studying the human gut microbiome into therapeutic strategies. Ultimately this review aims to discuss our current knowledge as well as highlight areas requiring further investigation.