Assembly of a unique membrane complex in type VI secretion systems of Bacteroidota.

The type VI secretion system (T6SS) of Gram-negative bacteria inhibits competitor cells through contact-dependent translocation of toxic effector proteins. In Proteobacteria, the T6SS is anchored to the cell envelope through a megadalton-sized membrane complex (MC). However, the genomes of Bacteroidota with T6SSs appear to lack genes encoding homologs of canonical MC components. Here, we identify five genes in Bacteroides fragilis (tssNQOPR) that are essential for T6SS function and encode a Bacteroidota-specific MC. We purify this complex, reveal its dimensions using electron microscopy, and identify a protein-protein interaction network underlying the assembly of the MC including the stoichiometry of the five TssNQOPR components. Protein TssN mediates the connection between the Bacteroidota MC and the conserved baseplate. Although MC gene content and organization varies across the phylum Bacteroidota, no MC homologs are detected outside of T6SS loci, suggesting ancient co-option and functional convergence with the non-homologous MC of Pseudomonadota.

Chlorate Contamination in Commercial Growth Media as a Source of Phenotypic Heterogeneity within Bacterial Populations.

Under anaerobic conditions, chlorate is reduced to chlorite, a cytotoxic compound that triggers oxidative stress within bacterial cultures. We previously found that BD Bacto Casamino Acids were contaminated with chlorate. In this study, we investigated whether chlorate contamination is detectable in other commercial culture media. We provide evidence that in addition to different batches of BD Bacto Casamino Acids, several commercial agar powders are contaminated with chlorate. A direct consequence of this contamination is that, during anaerobic growth, Escherichia coli cells activate the expression of msrP, a gene encoding periplasmic methionine sulfoxide reductase, which repairs oxidized protein-bound methionine. We further demonstrate that during aerobic growth, progressive oxygen depletion triggers msrP expression in a subpopulation of cells due to the presence of chlorate. Hence, we propose that chlorate contamination in commercial growth media is a source of phenotypic heterogeneity within bacterial populations. IMPORTANCE Agar is arguably the most utilized solidifying agent for microbiological media. In this study, we show that agar powders from different suppliers, as well as certain batches of BD Bacto Casamino Acids, contain significant levels of chlorate. We demonstrate that this contamination induces the expression of a methionine sulfoxide reductase, suggesting the presence of intracellular oxidative damage. Our results should alert the microbiology community to a pitfall in the cultivation of microorganisms under laboratory conditions.

Methionine oxidation in bacteria: A reversible post-translational modification.

Methionine is a sulfur-containing residue found in most proteins which are particularly susceptible to oxidation. Although methionine oxidation causes protein damage, it can in some cases activate protein function. Enzymatic systems reducing oxidized methionine have evolved in most bacterial species and methionine oxidation proves to be a reversible post-translational modification regulating protein activity. In this review, we inspect recent examples of methionine oxidation provoking protein loss and gain of function. We further speculate on the role of methionine oxidation as a multilayer endogenous antioxidant system and consider its potential consequences for bacterial virulence.

Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium.

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here, we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMHs) and show that conserved glutamate residues in GldL TMH2 are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane, suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

Cellular heterogeneity in DNA alkylation repair increases population genetic plasticity.

DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the adaptive response that increases the resistance of Escherichia coli cells to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are essentially absent. Whereas genetic deletion of these proteins causes extreme sensitivity to alkylation stress, a temporary lack of expression is tolerated and increases genetic plasticity of the whole population. We demonstrated this by monitoring the dynamics of nascent DNA mismatches during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. We propose that stochastic modulation of DNA repair capacity by the adaptive response creates a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.

Bacterial phenotypic heterogeneity in DNA repair and mutagenesis.

Genetically identical cells frequently exhibit striking heterogeneity in various phenotypic traits such as their morphology, growth rate, or gene expression. Such non-genetic diversity can help clonal bacterial populations overcome transient environmental challenges without compromising genome stability, while genetic change is required for long-term heritable adaptation. At the heart of the balance between genome stability and plasticity are the DNA repair pathways that shield DNA from lesions and reverse errors arising from the imperfect DNA replication machinery. In principle, phenotypic heterogeneity in the expression and activity of DNA repair pathways can modulate mutation rates in single cells and thus be a source of heritable genetic diversity, effectively reversing the genotype-to-phenotype dogma. Long-standing evidence for mutation rate heterogeneity comes from genetics experiments on cell populations, which are now complemented by direct measurements on individual living cells. These measurements are increasingly performed using fluorescence microscopy with a temporal and spatial resolution that enables localising, tracking, and counting proteins with single-molecule sensitivity. In this review, we discuss which molecular processes lead to phenotypic heterogeneity in DNA repair and consider the potential consequences on genome stability and dynamics in bacteria. We further inspect these concepts in the context of DNA damage and mutation induced by antibiotics.

Type IX secretion system PorM and gliding machinery GldM form arches spanning the periplasmic space.

Type IX secretion system (T9SS), exclusively present in the Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N (P. gingivalis) or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-Å length that span most of the periplasmic space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior.

Probing Inner Membrane Protein Topology by Proteolysis.

Inner membrane proteins are inserted into the membrane via α-helices. These helices do not only constitute membrane anchors but may mediate specific interactions with membrane protein partners or participate in energetic processes. The number, location, and orientation of these helices is referred to as topology. Bitopic membrane proteins that consist of a single membrane-embedded domain connecting two soluble domains are distinguished from polytopic ones that consist of multiple membrane-spanning helices connected by extramembrane domains. Defining inner membrane protein topology could be achieved by different methods. Here we describe a protease accessibility assay that makes it possible to define topology based on digestion profiles.

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex.

The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.