Biology subjects

Navarre, W. W.

Publications and source records attributed to Navarre, W. W..

8 recordsLinked to original sources

bioRxiv2026

Utilizing natural competence to genetically manipulate Lactobacillus iners

The healthy human vaginal microbiota is typically dominated by one species of Lactobacillus: L. iners, L. crispatus, L. jensenii, or L. gasseri. L. iners, the most prevalent vaginal microbe globally, is the most fastidious of the vaginal lactobacilli, has the smallest genome, and produces less lactic acid (only the L-isoform). L. iners is also less protective against bacterial vaginosis, and uniquely encodes a cholesterol-dependent cytolysin, inerolysin, suggesting it may be a pathobiont. Despite its central role in the health of over one billion females, L. iners biology remains poorly understood in part due to a lack of genetic editing tools. Here, we present findings that L. iners is naturally competent and can be transformed easily by exogenous DNA. Natural competence was leveraged to disrupt the iny gene encoding inerolysin, and comGA, encoding the ATPase component of the competence pilus. Both gene disruptions were accomplished using PCR assembled DNA fragments comprising a drug resistance gene cassette (tetM or ermB) flanked by [~]2 kb regions of homology to the L. iners chromosome. We further demonstrate that comGA is essential for L. iners transformation. The ability to rapidly perform targeted deletions in L. iners with in vitro generated DNA templates provides a straightforward and much needed method to probe the genetics and physiology of these important vaginal bacteria. IMPORTANCEThis study describes, to our knowledge, the first method for genetically manipulating L. iners, the most prevalent bacteria of the human vaginal microbiota. This work paves the way for the rapid development of genetic tools to explore L. iners physiology in the context of the vaginal microbiome and potentially alter its properties as a probiotic.

microbiology↗

bioRxiv2024

Practical Media Formulations for Rapid Growth of Lactobacillus iners and Other Vaginal Bacteria

Vaginal microbiome composition is closely tied to host health. A microbiome dominated by specific anaerobes (e.g., Gardnerella vaginalis) is termed bacterial vaginosis (BV) and is associated with negative health outcomes, while colonization by Lactobacillus species is thought to protect against BV. However, the role of the species Lactobacillus iners in vaginal health is controversial, with evidence that some strains may not protect against BV while others do. To better characterize L. iners strains, their interactions with vaginal bacteria and human cells need to be investigated in vitro, but this has been impeded by the lack of liquid media for rapid L. iners growth. We have developed three liquid media formulations for L. iners growth: Serradors Lactobacilli-adapted Iscoves Medium (SLIM) which leads to robust L. iners growth, a vaginally adapted version of SLIM (SLIM-V) and a chemically defined medium (SLIM-CD). SLIM and SLIM-V lead to dramatically improved L. iners growth compared to previously published formulations and support growth of other vaginal bacteria, including L. crispatus, L. jensenii, L. gasseri and G. vaginalis. SLIM-CD leads to slower growth but could prove useful for characterizing L. iners nutrient requirements or metabolite production. A modified version of SLIM-V supports growth of human cervical epithelial cells and provides a base for future co-culture work. Here, we present the formulations of SLIM, SLIM-V and SLIM-CD, and compare the growth of bacterial strains and human cells in the media.

microbiology↗

bioRxiv2024

Evolutionary and functional divergence of Sfx, a plasmid-encoded H-NS homolog, underlies the regulation of IncX plasmid conjugation

Conjugative plasmids are widespread among prokaryotes, highlighting their evolutionary success. Despite being an important route for plasmid transmission, most natural plasmids do not constitutively conjugate and are conjugatively repressed by default. In the F plasmid, the negative regulation of conjugation is partially mediated by the chromosomal nucleoid structuring protein (H-NS). Recent bioinformatic analyses have revealed that plasmid-encoded H-NS homologs are widespread and exhibit high sequence diversity. However, the functional roles of most of these homologs and the selective forces driving their phylogenetic diversification remain unclear. In this study, we characterized the functionality and evolution of Sfx, a H-NS homolog encoded by the model IncX2 plasmid R6K. We demonstrate that Sfx, but not chromosomal H-NS, can repress R6K conjugation. Notably, we find evidence of positive selection acting on the ancestral Sfx lineage. These positively selected sites are located in the dimerization, oligomerization, and DNA-binding interfaces, many of which contribute to R6K repression activity - indicating that adaptive evolution drove the functional divergence of Sfx. We additionally show that Sfx can physically interact with various chromosomally encoded proteins, including H-NS, StpA, and Hha. Hha enhances the ability of Sfx to regulate R6K conjugation, suggesting that Sfx retained functionally important interactions with chromosomal silencing proteins. Surprisingly, the loss of Sfx does not negatively affect the stability or dissemination of R6K in laboratory conditions, reflecting the complexity of selective pressures favoring conjugation repression. Overall, our study sheds light on the functional and evolutionary divergence of a plasmid-borne H-NS-like protein, highlighting how these loosely specific DNA-binding proteins evolved to specifically regulate different plasmid functions. IMPORTANCEConjugative plasmids play a crucial role in spreading antimicrobial resistance and virulence genes. Most natural conjugative plasmids conjugate only under specific conditions. Therefore, studying the molecular mechanisms underlying conjugation regulation is essential for understanding antimicrobial resistance and pathogen evolution. In this study, we characterized the conjugation regulation of the model IncX plasmid R6K. We discovered that Sfx, a H-NS homolog carried by the plasmid, represses conjugation. Molecular evolutionary analyses combined with gain-of-function experiments indicate that positive selection underlies the conjugation repression activity of Sfx. Additionally, we demonstrate that the loss of Sfx does not adversely affect R6K maintenance under laboratory conditions, suggesting additional selective forces favoring Sfx carriage. Overall, this work underscores the impact of protein diversification on plasmid biology, enhancing our understanding of how molecular evolution affects broader plasmid ecology.

microbiology↗

bioRxiv2022

Enhanced cultured diversity of the mouse gut microbiota enables custom-made synthetic communities

Microbiome research is hampered by the fact that many bacteria are still unknown and by the lack of publicly available isolates. Fundamental and clinical research is in need of comprehensive and well-curated repositories of cultured bacteria from the intestine of mammalian hosts. In this work, we expanded the mouse intestinal bacterial collection (www.dsmz.de/miBC) to 212 strains, all publicly available and taxonomically described. This includes the study of strain-level diversity, small-sized bacteria, and the isolation and characterization of the first cultured members of one novel family, 10 novel genera, and 39 novel species. We demonstrate the value of this collection by performing two studies. First, metagenome-educated design allowed establishing custom synthetic communities (SYNs) that reflect different susceptibilities to DSS-induced colitis. Second, nine phylogenetically and functionally diverse species were used to amend the Oligo-Mouse Microbiota (OMM)12 model [Brugiroux et al. 2016 Nat Microbiol]. These strains compensated for differences observed between gnotobiotic OMM12 and specific pathogen-free (SPF) mice at multiple levels, including body composition and immune cell populations (e.g., T-cell subtypes) in the intestine and associated lymphoid tissues. Ready-to-use OMM stocks are available to the community for use in future studies. In conclusion, this work improves our knowledge of gut microbiota diversity in mice and enables functional studies via the modular use of isolates.

microbiology↗

bioRxiv2019

Salmonella stress response mediates growth repression in succinate media

Bacteria have evolved to sense and respond to their environment by altering gene expression and metabolism to promote growth and survival. In this work we demonstrate that Salmonella displays an extensive (>30 hour) lag in growth when subcultured into media where dicarboxylates such as succinate are the sole carbon source. This growth lag is regulated in part by RpoS, the RssB anti-adaptor IraP, translation elongation factor P, and to a lesser degree the stringent response. We also show that small amounts of proline or citrate can trigger early growth in succinate media and that, at least for proline, this effect requires the multifunctional enzyme/regulator PutA. We demonstrate that activation of RpoS results in the repression of dctA, encoding the primary dicarboxylate importer, and that constitutive expression of dctA induced growth. This dicarboxylate growth lag phenotype is far more severe across multiple Salmonella isolates than in its close relative E. coli. Replacing 200 nt of the Salmonella dctA promoter region with that of E. coli was sufficient to eliminate the observed lag in growth. We hypothesize that this cis-regulatory divergence might be an adaptation to Salmonellas virulent lifestyle where levels of phagocyte-produced succinate increase in response to bacterial LPS. We found that impairing dctA repression had no effect on Salmonellas survival in acidified succinate or in macrophage but propose alternate hypotheses of fitness advantages acquired by repressing dicarboxylate uptake. ImportanceBacteria have evolved to sense and respond to their environment to maximize their chance of survival. By studying differences in the responses of pathogenic bacteria and closely related non-pathogens, we can gain insight into what environments they encounter inside of an infected host. Here we demonstrate that Salmonella diverges from its close relative E. coli in its response to dicarboxylates such as the metabolite succinate. We show that this is regulated by stress response proteins and ultimately can be attributed to Salmonella repressing its import of dicarboxylates. Understanding this phenomenon may reveal a novel aspect of the Salmonella virulence cycle, and our characterization of its regulation yields a number of mutant strains that can be used to further study it.

microbiology↗

bioRxiv2019

Salmonella ItaR responds to itaconate in macrophage

Itaconate is a dicarboxylic acid that inhibits the isocitrate lyase enzyme of the bacterial glyoxylate shunt. Activated macrophages have been shown to produce itaconate, suggesting that these immune cells may employ this metabolite as a weapon against invading bacteria. Here we demonstrate that, in vitro, itaconate can exhibit bactericidal effects under acidic conditions similar to the pH of a macrophage phagosome. In parallel, successful pathogens including Salmonella have acquired a genetic operon encoding itaconate degradation proteins, which are induced heavily in macrophages. We characterize the regulation of this operon by the neighbouring gene, ripR, in specific response to itaconate. Moreover, we develop an itaconate biosensor based on the operon promoter that can detect itaconate in a semi-quantitative manner and, when combined with the ripR gene, is sufficient for itaconate-regulated expression in E. coli. Using this biosensor with fluorescence microscopy, we observe bacteria responding to itaconate in the phagosomes of macrophage and provide additional evidence that interferon-{gamma} stimulates macrophage itaconate synthesis and that J774 mouse macrophages produce substantially more itaconate than the human THP-1 monocyte cell line. In summary, we examine the role of itaconate as an antibacterial metabolite in mouse and human macrophage, characterize the regulation of Salmonellas defense against it, and develop it as a convenient itaconate biosensor and inducible promoter system. ImportanceIn response to invading bacteria, immune cells can produce a molecule called itaconate, which can inhibit microbial metabolism. Here we show that itaconate can also directly kill Salmonella when combined with moderate acidity, further supporting itaconates role as an antibacterial weapon. We also discover how Salmonella recognizes itaconate and activates a defense to degrade it, and we harness this response to make a biosensor that detects the presence of itaconate. This biosensor is versatile, working in Salmonella enterica or lab strains of Escherichia coli, and can detect itaconate quantitatively in the environment and in immune cells. By understanding how immune cells kill bacteria and how the microbes defend themselves, we can better develop novel antibiotics to inhibit pathogens such as Salmonella.

microbiology↗

bioRxiv2019

Growth Phase Dependent Chromosome Condensation and H-NS Protein Redistribution in E. coli Under Osmotic Stress

The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in E. coli bacteria. We have applied super-resolved microscopy to quantify the intracellular, spatial reorganization of H-NS in response to osmotic stress. We find that H-NS shows a growth phase dependent response to hyperosmotic shock. In stationary phase, H-NS detaches from a tightly compacted bacterial chromosome and is excluded from the nucleoid volume over an extended period of time. This behaviour is absent during rapid growth but may be induced by exposing the osmotically stressed culture to the DNA gyrase inhibitor, coumermycin. This compaction/H-NS exclusion phenomenon occurs in the presence of either potassium or sodium ions and is independent of the stress responsive sigma factor RpoS, or the H-NS paralog StpA.

microbiology↗

bioRxiv2018

The Evolution of SlyA/RovA Transcription Factors From Repressors to Counter-Silencers in Enterobacteriaceae

Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally-responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently-transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to counter-silence horizontally-acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands.

microbiology↗