SigV mediates lysozyme resistance in Enterococcus faecalis via RsiV and PgdA

Enterococcus faecalis is a gut commensal but transitions to a pathogenic state as a consequence of intestinal dysbiosis and/or the presence of indwelling medical devices causing a wide range of infections. One of the unique features of E. faecalis is its ability to display high level resistance to lysozyme, an important host defense of the innate immune response. Lysozyme resistance in E. faecalis is known to be mediated by the e xtra c ytoplasmic f unction (ECF) sigma factor, SigV. PgdA and RsiV expression is directly regulated by SigV, but pgdA and rsiV mutants display nominal changes in lysozyme resistance, suggesting that additional gene products in the SigV regulon contribute to lysozyme resistance. Using RNA-seq analysis, we compared the transcriptional profile of the parental strain to an isogenic sigV mutant and show that apart from sigV , only rsiV and pgdA expression was induced upon lysozyme exposure. The combined deletion mutant of both rsiV and pgdA rendered E. faecalis sensitive to lysozyme at a level comparable to the sigV mutant, highlighting the limited SigV regulon. Several additional genes were also induced upon lysozyme exposure, but in a SigV-independent fashion. Overexpression of pgdA from a SigV-independent promoter restored lysozyme resistance in a sigV deletion mutant and also induced cell chaining. Overexpression of rsiV from a SigV-independent promoter only partially restored lysozyme resistance in a sigV mutant. Overall, we provide evidence for a simple adaptation to lysozyme stress, in which SigV controls the expression of rsiV and pgdA , and that both gene products contribute to lysozyme resistance. Importance Enterococcus faecalis causes healthcare-associated infections and displays resistance to a variety of antibiotics and molecules of the innate immune system. SigV has been shown to play an important role in enterococcal lysozyme resistance. Even though several proteins have been implicated in enterococcal lysozyme resistance, a complete SigV-dependent regulon has not been functionally characterized as being responsible for the dramatic increase in lysozyme susceptibility displayed by a sig V mutant. Using RNA-seq, we have identified the SigV regulon to be comprised of two gene loci, sigV - rsiV and pgdA . Deletion of both rsiV and pgdA renders E. faecalis susceptible to lysozyme on par with a sigV mutant. We also demonstrate that overproduction of rsiV and pgdA contribute to lysozyme resistance in susceptible strains.

4-Methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans

In bacteria, DNA methylation can be facilitated by ‘orphan’ DNA methyltransferases lacking cognate restriction endonucleases, but whether and how these enzymes control key cellular processes are poorly understood. The effects of a specific modification, 4-methylcytosine (4mC), are even less clear, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase from the understudied pathogen Leptospira spp. Inactivating this enzyme resulted in complete abrogation of CTAG motif methylation, leading to genome-wide dysregulation of gene expression. Mutants exhibited growth defects, decreased adhesion to host cells, higher susceptibility to LPS-targeting antibiotics, and, importantly, were no longer virulent in an acute infection model. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter of the gene. The genes that comprise the regulon of this sigma factor were, accordingly, dysregulated in the methylase mutant and in a strain overexpressing the sigma factor. Our results highlight the importance of 4mC in Leptospira physiology, and suggest the same of other understudied species.

Novel Tat-Dependent Protein Secretion

ABSTRACT The transcription initiation signal elicited by the binding of ferric citrate to the outer membrane FecA protein is transmitted by the FecR protein across the cytoplasmic membrane to the FecI extracytoplasmic function (ECF) sigma factor. In this issue of Journal of Bacteriology, I. J. Passmore, J. M. Dow, F. Coll, J. Cuccui, et al. (J Bacteriol 202:e00541-19, 2020, https://doi.org/10.1128/JB.00541-19) report that the FecR sequence contains both the twin-arginine signal motif and the secretory (Sec) avoidance motif typical of proteins secreted by the twin-arginine translocation (TAT) system. The same study shows that FecR is indeed secreted by Tat and represents a new class of bitopic Tat-dependent membrane proteins.

The ECF sigma factor PvdS regulates the type I-F CRISPR-Cas system in Pseudomonas aeruginosa

During infection, successful colonization of bacteria requires a fine-tuned supply of iron acquired via iron transport systems. However, the transport systems serve as phage attachment sites and entry portals for foreign nucleic acid. Most bacteria possess the CRISPR-Cas system, which targets and destroys foreign nucleic acids and prevents deleterious effects of horizontal gene transfer. To understand the regulation of the CRISPR-Cas system, we performed genome-wide random transposon mutagenesis which led to the identification of the Extracytoplasmic Function (ECF) Sigma factor, PvdS as a regulator of the Type I-F CRISPR-Cas system in P. aeruginosa. We show that under iron-depleted conditions PvdS induces the expression of the type I-F CRISPR-Cas system. This regulatory mechanism involves direct interaction of PvdS with specific binding sites in the promoter region of cas1. Furthermore, activation of the CRISPR-Cas system under iron-depleted conditions increases horizontal gene transfer (HGT) interference and adaptation. The PvdS activation of the CRISPR-Cas system under iron limitation highlights the versatility of the P. aeruginosa in multitasking its regulatory machinery to integrate multiple stress factors.ImportanceP. aeruginosa infects a wide range of host organisms and adapts to various environmental stress factors such as iron limitation due to its elaborate regulatory system. P aeruginosa possesses the type I-F CRISPR-Cas system as a defense mechanism against phages infection and HGT. This work highlights the ability of P. aeruginosa to multitask its iron regulatory system to control the CRISPR-Cas system under a physiologically relevant stress factor such as iron limitation where the bacteria are vulnerable to phage infection. It also adds to the knowledge of the regulation of the CRISPR-Cas system in bacteria and presents a possible target that could prevent the emergence of phage resistance via the CRISPR-Cas system during the development of phage therapy.

A quasi-integral controller for adaptation of genetic modules to variable ribosome demand

The behavior of genetic circuits is often poorly predictable. A gene’s expression level is not only determined by the intended regulators, but also largely dictated by changes in ribosome availability imparted by activation or repression of other genes. To address this problem, we design a quasi-integral biomolecular feedback controller that enables the expression level of any gene of interest (GOI) to adapt to changes in available ribosomes. The feedback is implemented through a synthetic small RNA (sRNA) that silences the GOI’s mRNA, and uses orthogonal extracytoplasmic function (ECF) sigma factor to sense the GOI’s translation and to actuate sRNA transcription. Without the controller, the expression level of the GOI is reduced by 50% when a resource competitor is activated. With the controller, by contrast, gene expression level is practically unaffected by the competitor. This feedback controller allows adaptation of genetic modules to variable ribosome demand and thus aids modular construction of complicated circuits.

A genomic island of Streptomyces coelicolor with the self-contained regulon of an ECF sigma factor

Streptomycetes constitute the largest genus of actinobacteria, living predominantly in soil and decaying vegetation. The bacteria are widely known for their filamentous morphologies and their capacity to synthesize antibiotics and other biologically active molecules. More than a decade ago, we and others identified 22 genomic islands thatStreptomyces coelicolorM145 possesses and otherStreptomycesstrains lack. One of these genomic islands, Genomic Island (GI) 6, encodes an extracytoplasmic function (ECF) sigma factor that we were characterizing in separate work. Here we report that artificial induction of the ECF sigma factor, which is encoded by SCO3450, causes the transcription of approximately one-fourth of GI 6, or ~26 mostly contiguous genes, to increase. More than half of the regulon encodes putative enzymes involved in small molecule metabolism. A putative haloacid dehalogenase is present. Genes encoding two putative anti-sigma factors flank SCO3450, the three genes residing within the regulon. Our data suggest that the ECF sigma factor and its regulon are a self-contained transcriptional unit that can be transferred by horizontal gene transfer. To our knowledge, only one other example has been identified of an ECF sigma factor and its contiguous regulon appearing to be transferrable by horizontal gene transfer [18,19]. Because the regulon appears not to be induced by the 44 growth conditions recently examined by Byung-Kwan Cho and colleagues [20], if it confers fitness toS. coelicolor, the regulon likely does so in as-yet unknown situations. Those situations might range from scavenging to detoxification to even communication within microbial communities.IMPORTANCEStreptomycesbacteria grow as hyphae that colonize soil and differentiate into spores when nutrients become scarce. In their terrestrial habitats, the bacteria encounter diverse conditions. Presumably so that the bacteria can cope with those conditions, the chromosomes of streptomycetes are highly dynamic, varying greatly in structure not only between species but also between closely related strains of a single species. The bacteria also have large numbers of extracytoplasmic function (ECF) sigma factors, which undoubtedly help the microorganisms respond to the plethora of challenges coming from the environment. This work illustrates these two threads ofStreptomycesbiology dovetailing: Genetic adaptability through horizontal gene transfer seems to have enabledStreptomyces coelicolorto acquire a self-contained transcriptional unit that consists of an ECF sigma factor and its regulon. The suggested facile movement of the regulon between microbial hosts indicates the value of the metabolism of small molecules possibly mediated by the regulon.