Diverse functions for acyltransferase-3 proteins in the modification of bacterial cell surfaces.

The acylation of sugars, most commonly via acetylation, is a widely used mechanism in bacteria that uses a simple chemical modification to confer useful traits. For structures like lipopolysaccharide, capsule and peptidoglycan, that function outside of the cytoplasm, their acylation during export or post-synthesis requires transport of an activated acyl group across the membrane. In bacteria this function is most commonly linked to a family of integral membrane proteins - acyltransferase-3 (AT3). Numerous studies examining production of diverse extracytoplasmic sugar-containing structures have identified roles for these proteins in O-acylation. Many of the phenotypes conferred by the action of AT3 proteins influence host colonisation and environmental survival, as well as controlling the properties of biotechnologically important polysaccharides and the modification of antibiotics and antitumour drugs by Actinobacteria. Herein we present the first systematic review, to our knowledge, of the functions of bacterial AT3 proteins, revealing an important protein family involved in a plethora of systems of importance to bacterial function that is still relatively poorly understood at the mechanistic level. By defining and comparing this set of functions we draw out common themes in the structure and mechanism of this fascinating family of membrane-bound enzymes, which, due to their role in host colonisation in many pathogens, could offer novel targets for the development of antimicrobials.

A BTP1 prophage gene present in invasive non-typhoidal Salmonella determines composition and length of the O-antigen of the lipopolysaccharide.

Salmonella Typhimurium isolate D23580 represents a recently identified ST313 lineage of invasive non-typhoidal Salmonellae (iNTS). One of the differences between this lineage and other non-iNTS S. Typhimurium isolates is the presence of prophage BTP1. This prophage encodes a gtrC gene, implicated in O-antigen modification. GtrC(BTP) (1) is essential for maintaining O-antigen length in isolate D23580, since a gtr(BTP) (1) mutant yields a short O-antigen. This phenotype can be complemented by gtrC(BTP) (1) or very closely related gtrC genes. The short O-antigen of the gtr(BTP) (1) mutant was also compensated by deletion of the BTP1 phage tailspike gene in the D23580 chromosome. This tailspike protein has a putative endorhamnosidase domain and thus may mediate O-antigen cleavage. Expression of the gtrC(BTP) (1) gene is, in contrast to expression of many other gtr operons, not subject to phase variation and transcriptional analysis suggests that gtrC is produced under a variety of conditions. Additionally, GtrC(BTP) (1) expression is necessary and sufficient to provide protection against BTP1 phage infection of an otherwise susceptible strain. These data are consistent with a model in which GtrC(BTP) (1) mediates modification of the BTP1 phage O-antigen receptor in lysogenic D23580, and thereby prevents superinfection by itself and other phage that uses the same O-antigen co-receptor.

CdiA promotes receptor-independent intercellular adhesion.

CdiB/CdiA proteins mediate inter-bacterial competition in a process termed contact-dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI(+) cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto-aggregation and biofilm formation in several species, but the contribution of CdiA-receptor interactions to these multi-cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA-BamA mediated cell-cell adhesion. However, CdiA also promotes auto-aggregation in a BamA-independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA-independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA-CdiA interactions. The BamA-dependent and BamA-independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth-inhibition activity. This adhesion should enable 'greenbeard' discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind-selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer.

Horizontally acquired glycosyltransferase operons drive salmonellae lipopolysaccharide diversity.

The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions.

Control of gene expression at a bacterial leader RNA, the agn43 gene encoding outer membrane protein Ag43 of Escherichia coli.

The family of agn alleles in Escherichia coli pathovars encodes autotransporters that have been implicated in biofilm formation, autoaggregation, and attachment to cells. The alleles all have long leader RNAs preceding the Ag43 translation initiation codon. Here we present an analysis of the agn43 leader RNA from E. coli K-12. We demonstrate the presence of a rho-independent transcription terminator just 28 bp upstream of the main translation start codon and show that it is functional in vitro. Our data indicate that an as-yet-unknown mechanism of antitermination of transcription must be operative in earlier phases of growth. However, as bacterial cell cultures mature, progressively fewer transcripts are able to bypass this terminator. In the K-12 leader sequence, two in-frame translation initiation codons have been identified, one upstream and the other downstream of the transcription terminator. For optimal agn43 expression, both codons need to be present. Translation from the upstream start codon leads to increased downstream agn43 expression. Our findings have revealed two novel modes of regulation of agn43 expression in the leader RNA in addition to the previously well-characterized regulation of phase variation at the agn43 promoter.

A novel fold for acyltransferase-3 (AT3) proteins provides a framework for transmembrane acyl-group transfer.

Acylation of diverse carbohydrates occurs across all domains of life and can be catalysed by proteins with a membrane bound acyltransferase-3 (AT3) domain (PF01757). In bacteria, these proteins are essential in processes including symbiosis, resistance to viruses and antimicrobials, and biosynthesis of antibiotics, yet their structure and mechanism are largely unknown. In this study, evolutionary co-variance analysis was used to build a computational model of the structure of a bacterial O-antigen modifying acetyltransferase, OafB. The resulting structure exhibited a novel fold for the AT3 domain, which molecular dynamics simulations demonstrated is stable in the membrane. The AT3 domain contains 10 transmembrane helices arranged to form a large cytoplasmic cavity lined by residues known to be essential for function. Further molecular dynamics simulations support a model where the acyl-coA donor spans the membrane through accessing a pore created by movement of an important loop capping the inner cavity, enabling OafB to present the acetyl group close to the likely catalytic resides on the extracytoplasmic surface. Limited but important interactions with the fused SGNH domain in OafB are identified, and modelling suggests this domain is mobile and can both accept acyl-groups from the AT3 and then reach beyond the membrane to reach acceptor substrates. Together this new general model of AT3 function provides a framework for the development of inhibitors that could abrogate critical functions of bacterial pathogens.

The fatty membrane that surrounds cells is an essential feature of all living things. It is a selective barrier, only allowing certain substances to enter and exit the cell, and it contains the proteins and carbohydrates that the cell uses to interact with its environment. In bacteria, the carbohydrates on the outer side of the membrane can become 'tagged' or modified with small chemical entities which often prove useful for the cell. Acyl groups, for example, allow disease-causing bacteria to evade the immune system and contribute to infections persisting in the body. As a rule, activated acyl groups are only found inside the cell, so they need to move across the membrane before they can be attached onto the carbohydrates at the surface. This transfer is performed by a group of proteins that sit within the membrane called the acyltransferase-3 (AT3) family. The structure of these proteins and the mechanism by which they facilitate membrane crossing have remained unclear. Newman, Tindall et al. combined computational and structural modelling techniques with existing experimental data to establish how this family of proteins moves acyl groups across the membrane. They focused on OafB, an AT3 protein from the foodborne bacterial pathogen Salmonella typhimurium. The experimental data used by the team included information about which parts of OafB are necessary for this protein to acylate carbohydrates molecules. In their experiments, Newman, Tindall et al. studied how different parts of OafB move, how they interact with the molecules that carry an acyl group to the membrane, and how the acyl group is then transferred to the carbohydrate acceptor. Their results suggest that AT3 family proteins have a central pore or hole, plugged by a loop. This loop moves and therefore 'unplug' the pore, resulting in the emergence of a channel across the membrane. This channel can accommodate the acyl-donating molecule, presenting the acyl group to the outer surface of the membrane where it can be transferred to the acceptor carbohydrate. The AT3 family of proteins participates in many cellular processes involving the membrane, and a range of bacterial pathogens rely on these proteins to successfully infect human hosts. The results of Newman Tindall et al. could therefore be used across the biological sciences to provide more detailed understanding of the membrane, and to inform the design of drugs to fight bacterial diseases.

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

Establishing and maintaining sequestration of Dam target sites for phase variation of agn43 in Escherichia coli.

Phase variation of the outer membrane protein Ag43 encoded by agn43 in Escherichia coli is controlled by an epigenetic mechanism. Sequestration of the regulatory region from Dam-dependent methylation has to be established and maintained throughout a generation to obtain and maintain the OFF phase. This work shows that hemimethylated DNA, which is formed by the passage of the DNA replication fork in an ON-phase cell, can be sequestered from methylation by OxyR binding, which is thus a key event for the switch from ON to OFF. No evidence was found that the protein SeqA, which also binds to the region, is involved in sequestration. To facilitate the dissection of this process further, a novel approach was introduced that does not alter the sequence of the regulatory region or the cellular concentration of Dam or OxyR, which consists of inserting auxiliary OxyR binding sites upstream of the regulatory region. Using this strategy, it was shown that the ON-to-OFF switch frequency can be modulated without changing the OFF-to-ON frequency. The data support a model in which in an ON-phase cell, the subcellular OxyR availability at the replication fork as it passes through the agn43 regulatory region is key for initiating an ON-to-OFF switch. In contrast, this availability is not a determining factor for the switch from OFF to ON. This finding shows that different variables affect these two stochastic events. This provides new insight into the events determining the stochastic nature of epigenetic phase variation.

Acetylation of Surface Carbohydrates in Bacterial Pathogens Requires Coordinated Action of a Two-Domain Membrane-Bound Acyltransferase.

Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an "AT3-SGNH fused" domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates.IMPORTANCE Acyltransferase-3 (AT3) domain-containing membrane proteins are involved in O-acetylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to antimicrobials, and biosynthesis of antibiotics. Their mechanism of action, however, is poorly characterized. We analyzed two acetyltransferases as models for this important family of membrane proteins, which modify carbohydrates on the surface of the pathogen Salmonella enterica, affecting immunogenicity, virulence, and bacteriophage resistance. We show that when these AT3 domains are fused to a periplasmic partner domain, both domains are required for substrate acetylation. The data show conserved elements in the AT3 domain and unique structural features of the periplasmic domain. Our data provide a working model to probe the mechanism and function of the diverse and important members of the widespread AT3 protein family, which are required for biologically significant modifications of cell-surface carbohydrates.

Spatial Organization of Expanding Bacterial Colonies Is Affected by Contact-Dependent Growth Inhibition.

Identifying how microbes are able to manipulate, survive, and thrive in complex multispecies communities has expanded our understanding of how microbial ecosystems impact human health and the environment. The ability of bacteria to negatively affect neighbors, through explicit toxin delivery systems, provides them with an opportunity to manipulate the composition of growing microbial communities. Contact-dependent inhibition (CDI) systems (a Type Vb secretion system) are a distinct subset of competition systems whose contribution to shaping the development of spatially structured bacterial communities are yet to be fully understood. Here, we compare the impact of different CDI systems, at both the single-cell and population level, to determine the key drivers of CDI-mediated competition within spatially structured bacterial populations. Through an iterative approach using both an Escherichia coli experimental system and computational modeling, we show that CDI systems have subtle and system-specific effects at the single-cell level, generating single-cell-wide boundaries between CDI-expressing inhibitor cells and their neighboring targets. Despite the subtle effects of CDI at a single-cell level, CDI systems greatly diminished the ability of susceptible targets to expand their range during colony growth. The inoculum density of the population, together with the CDI system-specific variables of the speed of inhibition after contact and biological cost of CDI, strongly affects CDI-mediated competition. In contrast, the magnitude of the toxin-induced growth retardation of target cells only weakly impacts the composition of the population. Our work reveals how distinct CDI systems can differentially affect the composition and spatial arrangement of bacterial populations.

Re-examining the role and random nature of phase variation.

Phase variation in bacteria is often considered a random process that has evolved to facilitate immune evasion in a host. Here, alternative biological roles for this process are presented and discussed, incorporating recent studies on nonpathogenic and commensal bacterial species. Furthermore, the integration of phase variation into bacterial regulatory networks and the relevance of this for considering phase variation as a random process are reviewed. Novel approaches are needed to study phase variation and its biological roles, but the insights obtained can contribute significantly to our understanding of the dynamic behaviour of bacterial populations and their interactions with the environment.

Spatial Dependence of DNA Damage in Bacteria due to Low-Temperature Plasma Application as Assessed at the Single Cell Level.

Low temperature plasmas (LTPs) generate a cocktail of reactive nitrogen and oxygen species (RNOS) with bactericidal activity. The RNOS however are spatially unevenly distributed in the plasma. Here we test the hypothesis that this distribution will affect the mechanisms underpinning plasma bactericidal activity focussing on the level of DNA damage in situ. For the first time, a quantitative, single cell approach was applied to assess the level of DNA damage in bacteria as a function of the radial distance from the centre of the plasma jet. Salmonella enterica on a solid, dry surface was treated with two types of LTP: an atmospheric-pressure dielectric barrier discharge plasma jet (charged and neutral species) and a radio-frequency atmospheric-pressure plasma jet (neutral species). In both cases, there was an inverse correlation between the degree of DNA damage and the radial distance from the centre of the plasma, with the highest DNA damage occurring directly under the plasma. This trend was also observed with Staphylococcus aureus. LTP-generated UV radiation was eliminated as a contributing factor. Thus valuable mechanistic information can be obtained from assays on biological material, which can inform the development of LTP as a complementary or alternative therapy for (topical) bacterial infections.

Phase variation: how to create and coordinate population diversity.

Phase variation yields phenotypic heterogeneity in a clonal population as the result of one of a limited number of known molecular mechanisms. These include slipped strand mispairing, site-specific recombination and epigenetic regulation mediated by DNA methylation. Recently new regulatory variants utilizing these mechanisms have been identified, which is facilitating the identification of additional phase variation events solely from genome sequence analysis. Furthermore, it is becoming increasingly clear that in many cases phase variation control is integrated with regulatory networks and with cellular processes of a growing cell. This review focuses specifically on these recent advances in the understanding of the regulation of phase variation.

Regulation and function of Ag43 (flu).

Antigen 43 (Ag43) is an abundant outer membrane protein in Escherichia coli belonging to the autotransporter family. Structure-function relationships of Ag43 proposed on the basis of experimental work and in silico analysis are discussed in context of insights derived from molecular modeling. New sequence analysis sheds light on the phylogeny of the allelic variants of the Ag43-encoding gene and identifies two distinct families that appear to be distributed between specific pathogenic and commensal isolates. The molecular mechanism that controls expression by phase variation to create population heterogeneity is discussed. Proposed roles of Ag43 expression for E. coli are summarized and the studies are put into perspective regarding the role of allelic variants, genetic background of the bacterial strain, and control of expression by phase variation. We conclude that future studies need to take into account these variables to obtain a complete understanding of the contribution of Ag43 expression to E. coli biology.

Slipped-strand mispairing can function as a phase variation mechanism in Escherichia coli.

Slipped-strand mispairing (SSM) has not been identified as a mechanism of phase variation in Escherichia coli. Using a reporter gene, we show that sequences that cause phase variation by SSM in Haemophilus influenzae also lead to phase variation when introduced onto the chromosome of E. coli, and the frequencies of switching are in the biologically relevant range. Thus, the absence of SSM-mediated phase variation in E. coli does not appear to be due to a mechanistic constraint.

Phase and antigenic variation in bacteria.

Phase and antigenic variation result in a heterogenic phenotype of a clonal bacterial population, in which individual cells either express the phase-variable protein(s) or not, or express one of multiple antigenic forms of the protein, respectively. This form of regulation has been identified mainly, but by no means exclusively, for a wide variety of surface structures in animal pathogens and is implicated as a virulence strategy. This review provides an overview of the many bacterial proteins and structures that are under the control of phase or antigenic variation. The context is mainly within the role of the proteins and variation for pathogenesis, which reflects the main body of literature. The occurrence of phase variation in expression of genes not readily recognizable as virulence factors is highlighted as well, to illustrate that our current knowledge is incomplete. From recent genome sequence analysis, it has become clear that phase variation may be more widespread than is currently recognized, and a brief discussion is included to show how genome sequence analysis can provide novel information, as well as its limitations. The current state of knowledge of the molecular mechanisms leading to phase variation and antigenic variation are reviewed, and the way in which these mechanisms form part of the general regulatory network of the cell is addressed. Arguments both for and against a role of phase and antigenic variation in immune evasion are presented and put into new perspective by distinguishing between a role in bacterial persistence in a host and a role in facilitating evasion of cross-immunity. Finally, examples are presented to illustrate that phase-variable gene expression should be taken into account in the development of diagnostic assays and in the interpretation of experimental results and epidemiological studies.