Biology subjects

Crosson, S.

Publications and source records attributed to Crosson, S..

At least 19 recordsLinked to original sources

bioRxiv2026

A Caulobacter hub taxon in the soybean root endosphere has a distinctive biofilm and engages in riboflavin-mediated mutualism with Bradyrhizobium

While it is well established that communities of microbes colonize the surfaces of plants as epiphytes and the interior plant tissues as endophytes, their ecological roles and colonization mechanisms remain under characterized. Members of the genus Caulobacter have emerged as hub taxa in epiphytic and endophytic microbiomes of diverse plant hosts, but the factors that support their colonization and community interactions have not been defined. Here, we characterize Caulobacter sp. RL271, a non-canonical member of the genus recently identified as a hub taxon in the soybean root endosphere. We demonstrate that the surface attachment strategies and biofilm architecture of Caulobacter sp. RL271 are determined by a matrix of capsular polysaccharide and cellulose rather than a polar holdfast adhesin, a classical defining feature of the genus. Both polysaccharide components influence the kinetics of plant root colonization in the model plant Arabidopsis thaliana. We further demonstrate that the ecology of Caulobacter sp. RL271 is shaped by its nutritional dependence on exogenous riboflavin and by interactions with Bradyrhizobium diazoefficiens, the nitrogen-fixing symbiont of soybean roots. This work advances understanding of Caulobacter biology and establishes RL271 as a tractable model for dissecting the functional role of hub taxa in root endophyte communities.

microbiology↗

bioRxiv2026

BR-bodies link RNA homeostasis to stress tolerance and intracellular fitness in Brucella

Bacterial gene expression depends on the coordinated regulation of RNA synthesis, processing, and decay. The conserved RNA degradosome scaffold, Ribonuclease E (RNase E), assembles into phase-separated bacterial ribonucleoprotein bodies (BR-bodies) through its C-terminal intrinsically disordered region (IDR). This IDR also scaffolds recruitment of degradosome client proteins that carry out post-transcriptional gene regulation. Whether BR-bodies regulate virulence programs to support infection, however, is largely undefined. We show that RNase E of the intracellular pathogen Brucella ovis forms RNA-dependent condensates in vivo and phase-separates with RNA in vitro, with the IDR necessary and sufficient for BR-body assembly. Deleting the IDR (rne({Delta}IDR)) did not impair growth but sensitized Brucella to host-relevant oxidative and cell-envelope stressors. Transcriptome-wide profiling that simultaneously resolved mRNA decay and processing revealed that BR-bodies primarily accelerate mRNA turnover while stabilizing a distinct subset of transcripts. Notably, BR-bodies control the processing and levels of virB type IV secretion system (T4SS) mRNA and the turnover of its key activators, linking condensate-based RNA regulation to a core virulence pathway. Consistent with virB dysregulation, the rne({Delta}IDR) mutant was severely attenuated in mammalian macrophages. To test whether phase separation is sufficient for BR-body function, we replaced the B. ovis IDR with the highly divergent Caulobacter crescentus IDR. This chimera assembled BR-bodies but rescued fitness incompletely, fully restoring oxidative-stress resistance but not cell-envelope stress resistance or intracellular fitness. BR-bodies therefore link RNA metabolism to Brucella stress resistance and infection, and their full function requires both phase separation and additional native IDR-specific activities such as degradosome interactions. ImportanceBiomolecular condensates are non-membrane bound organelles that organize biological processes across all domains of life, but their role in bacterial infection is not well characterized. We show that Brucella, an intracellular bacterial pathogen and causative agent of the disease brucellosis, relies on biomolecular condensates called BR-bodies to control RNA stability and regulate gene expression. Without BR-bodies, Brucella is sensitized to host-relevant chemical stresses, cannot properly regulate essential infection machinery, and has severely diminished fitness within mammalian host cells. These results indicate that control of RNA levels by biomolecular condensates is required for Brucella survival within animal hosts, providing evidence that phase separation is a fundamental mechanism underlying Brucella adaptation to the hostile environment encountered during infection.

microbiology↗

bioRxiv2026

A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus burden at 3 h post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support early infection by B. abortus and B. ovis without impairing general phagocytosis. Gene set enrichment analysis further revealed positive regulators of mTORC1 signaling as key host factors; this result was validated through targeted disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Thus, the AKT-Ragulator-mTORC1 signaling axis contributes to the establishment of a permissive intracellular niche during early Brucella infection. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that support early Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.

microbiology↗

bioRxiv2025

An N4-like Caulobacter phage requires host smooth lipopolysaccharide biosynthesis for infection

Caulobacter species are Alphaproteobacteria that commonly inhabit plant-associated and aquatic microbial communities. Although Caulobacter is widespread and has long served as a model for the study of bacterial cell biology, our understanding of the diversity of viruses that infect Caulobacter species is limited. Here, we describe the discovery and characterization of Circe, a freshwater N4-like podophage belonging to the Schitoviridae family that infects C. crescentus. We isolated two variants, CirceC and CirceH, that differ by a single nucleotide resulting in an F91I substitution in Gp063, an uncharacterized protein found in diverse bacteriophages and bacteria. While both Circe variants adsorb to C. crescentus with similar efficiency, they produce morphologically distinct plaques and display different infection dynamics. Through forward genetic selection and genome-wide transposon fitness profiling, we identified C. crescentus genes involved in cell envelope assembly, membrane sphingolipid biosynthesis, and envelope polysaccharide biosynthesis that influence susceptibility to Circe infection. Loss-of-function mutations in a predicted nucleoside diphosphate sugar epimerase and multiple genes required for smooth lipopolysaccharide (S-LPS) biosynthesis and export conferred strong resistance to infection. These results support a model in which S-LPS functions as a receptor for phage Circe. Our study expands the known repertoire of Caulobacter phages and adds to a growing understanding of the role of envelope polysaccharides in bacterial infection by N4-family phages. IMPORTANCEViruses that infect bacteria and archaea, known as phages, shape microbial community structure and function. Phages initiate infection by binding to specific molecules on the surface of host cells. Yet for many microbes, the identities of infecting phages and their corresponding host receptors remain poorly defined. Caulobacter spp. are ecologically important bacteria that can produce a variety of protein surface structures, including pili, a flagellum, and a surface layer (S-layer), all of which have been identified as phage receptors in this genus. We discovered Caulobacter phage Circe and provide evidence that it relies on host smooth lipopolysaccharide to infect C. crescentus. This study broadens understanding of phage-host interactions in Caulobacter and establishes Circe as a new system to investigate the molecular mechanisms by which phages engage with bacterial cells.

microbiology↗

bioRxiv2025

The hiuABC Operon Mediates Xenosiderophore Utilization in Caulobacter crescentus

Caulobacter species are common residents of soil and aquatic ecosystems, where bioavailable iron is often extremely limited. Like other diderm bacteria, C. crescentus can acquire Fe(III) via outer-membrane TonB-dependent transporters (TBDTs) that recognize and import ferric siderophore complexes. Although C. crescentus is not known to synthesize siderophores, it encodes multiple TBDTs that are transcriptionally regulated by the ferric uptake repressor (Fur), suggesting it acquires iron by scavenging xenosiderophores produced by neighboring microbes. To identify C. crescentus genes required for xenosiderophore utilization, we developed a barcoded transposon screen using ferrioxamine B (FXB), a hydroxamate-family siderophore produced by soil actinomycetes, as a model substrate. This screen identified hiuABC, a conserved, Fur-regulated operon that supports FXB-dependent iron acquisition. We provide evidence that hiuA encodes the primary TBDT responsible for uptake of ferrioxamines and ferrichrome (FC), structurally distinct members of the hydroxamate siderophore family. hiuB encodes a PepSY-domain protein with structural similarity to Pseudomonas aeruginosa FoxB, a known periplasmic ferri-siderophore reductase. hiuC encodes a small, hypothetical membrane protein predicted to form a functional complex with HiuB in the inner membrane. Both hiuB and hiuC are required for utilization of FXB and ferrioxamine E (FXE), indicating a shared role in iron acquisition from ferrioxamines. Surprisingly, utilization of FC as an iron source required hiuB but not hiuC, suggesting a substrate-specific role for HiuC in ferri-siderophore processing. We conclude that the conserved hiuABC operon encodes a set of proteins that enable bacteria to acquire iron from structurally diverse hydroxamate-family siderophores. ImportanceIron is often a limiting nutrient due to its poor solubility in the presence of oxygen. To overcome this, some microbes produce specialized molecules known as siderophores, which tightly bind and solubilize iron, facilitating its uptake into the cell. Caulobacter species are common in freshwater, marine, and soil environments, and there is emerging evidence they play important roles in plant-associated microbial communities. Here we report the discovery of a three-gene system that allows C. crescentus to acquire iron from a set of siderophores produced by select soil bacteria and fungi. We define functional roles for each protein component of this system, which informs a mechanism by which Caulobacter can pirate iron-scavenging molecules produced by its neighbors.

microbiology↗

bioRxiv2025

Non-redundant cardiolipin synthases support membrane integrity and stress resilience in Bacteroides fragilis

Gut-resident bacteria must tolerate diverse membrane-disrupting agents, including bile acids, to maintain colonization. Cardiolipin is an anionic phospholipid that supports membrane integrity and stress resilience in many bacteria. However, cardiolipin synthases remain poorly characterized in the Bacteroidota, a dominant phylum of the human gut microbiota. The prevalent gut commensal Bacteroides fragilis encodes two predicted cardiolipin synthases, ClsA and ClsB. We previously identified both cls genes as bile-acid fitness factors in B. fragilis P207, but the individual contributions of ClsA and ClsB to cell physiology had remained undefined. Here we combine targeted gene deletion with high-resolution lipidomics, metabolomics, and elemental mass spectrometry to show that the two enzymes have non-redundant functions in the cell. Cardiolipin is a minor lipid in the B. fragilis membrane, and both Cls enzymes contribute to its production. clsA and clsB differ in growth-phase expression, in their effects on cell morphology, and in their associated cardiolipin species. Loss of each enzyme also produces distinct changes in fitness under several membrane-perturbing stresses and in the broader cellular metabolome, including compound classes with documented bioactivity in mammalian hosts. In contrast to the acute ion-gradient disruption caused by the secondary bile acid deoxycholate, deletion of both synthases did not measurably alter steady-state intracellular ion levels under standard growth conditions, indicating that cardiolipin loss does not perturb basal ion homeostasis under these conditions. Together, these results define non-redundant roles for two cardiolipin synthases in a common member of the human gut microbiota. ImportanceInflammatory bowel diseases affect millions of people worldwide. The gut bacterium Bacteroides fragilis is a normal member of the human intestinal microbiota that can also bloom to high abundance in inflamed guts. To survive within the gut, B. fragilis must maintain the integrity and function of its cell membrane. In this study, we characterize the functional role of two B. fragilis genes that contribute to the synthesis of the membrane lipid, cardiolipin. We find that the two cardiolipin synthase genes are not functionally interchangeable; each impacts the cells lipid pool in distinct ways and contributes differently to how B. fragilis responds to membrane stress. Our work provides insight into how a common gut bacterium adapts to conditions encountered in the intestine, and improves understanding of membrane biology in this important group of gut microbes.

microbiology↗

bioRxiv2025

Reversion of a RND transporter pseudogene uncovers latent stress resistance in Brucella ovis

Small-molecule screens can advance therapeutic discovery while yielding new insights into pathogen biology. Through a luminescence-based screen, we identified clinically approved dihydropyridines that reduced the fitness of the intracellular pathogen Brucella ovis within mammalian phagocytes. Given the established role of dihydropyridines as inhibitors of mammalian L-type calcium channels and our observation that drug treatment perturbed calcium and manganese levels in host phagocytes, we initially hypothesized a host-directed mechanism of action. However, dose-response assays in axenic medium revealed that these drugs can directly inhibit B. ovis growth. To investigate the genetic basis of B. ovis susceptibility to dihydropyridine treatment, we selected for mutants capable of growing in the presence of cilnidipine. Cilnidipine-resistant isolates carried single-base deletions in the bepE pseudogene that restored an open reading frame encoding an RND-family transporter subunit. B. ovis is an ovine venereal pathogen that has experienced significant pseudogenization in its recent evolutionary history. Frameshift mutations that restored bepE function in B. ovis increased its resistance not only to dihydropyridines but also to a broad range of membrane-disrupting agents, including bile acid. Conversely, deleting bepE in Brucella abortus, a related zoonotic species that retains an intact version of the gene, increased its sensitivity to bile acid in vitro and to cilnidipine in the intracellular niche. We conclude that bepE is a key determinant of chemical stress resistance in Brucella spp., and that its pseudogenization in B. ovis contributes to the documented hypersensitivity of this host-restricted lineage to chemical stressors. Author SummaryBrucella species are intracellular bacterial pathogens that infect diverse wildlife and livestock and can also cause disease in humans. We developed a high-throughput assay to identify compounds that reduce the intracellular fitness of Brucella ovis, a globally distributed pathogen of sheep. This screen identified two dihydropyridine-class hypertension drugs as inhibitors of B. ovis fitness within mammalian cells. While dihydropyridines are established inhibitors of calcium channels in mammals, our results revealed that they also directly inhibited B. ovis growth. The B. ovis genome contains over 300 pseudogenes, and we discovered that resistance to dihydropyridines and other chemical stressors can emerge through frameshift mutations that resurrect the function of bepE, a conserved pseudogene encoding a membrane transporter subunit. In other Brucella species, bepE remains intact, and we show that deleting this gene in the zoonotic pathogen Brucella abortus increases susceptibility to both dihydropyridine treatment and membrane-disrupting stress. Our study provides evidence that pseudogenes like bepE can serve as a latent reservoir of adaptive functions in B. ovis, including drug and stress resistance.

microbiology↗

bioRxiv2024

A co-conserved gene pair supports Caulobacter iron homeostasis during chelation stress

Synthetic metal chelators are widely used in industrial, clinical, and agricultural settings, leading to their accumulation in the environment. We measured the growth of Caulobacter crescentus, a soil and aquatic bacterium, in the presence of the ubiquitous chelator ethylenediaminetetraacetic acid (EDTA) and found that it restricts growth by lowering intracellular iron levels. Using barcoded transposon sequencing, we identified an operonic gene pair, cciT-cciO, that is required to maintain iron homeostasis in laboratory media during EDTA challenge. cciT encodes one of four TonB-dependent transporters that are regulated by the ferric uptake repressor (Fur) and stands out among this group of genes in its ability to support Caulobacter growth across diverse media conditions. The function of CciT strictly requires cciO, which encodes a cytoplasmic FeII dioxygenase-family protein. Our results thus define a functional partnership between an outer membrane iron receptor and a cytoplasmic dioxygenase that are broadly co-conserved in Proteobacteria. We expanded our analysis to natural environments by examining the growth of mutant strains in freshwater from two lakes, each with biochemical and geochemical profiles that differ markedly from standard laboratory media. In lake water, Caulobacter growth did not require cciT or cciO and was less affected by EDTA treatment. This result aligns with our observation that EDTA toxicity is influenced by common forms of biologically chelated iron and the spectrum of free cations present in the medium. Our study defines a conserved iron acquisition system in Proteobacteria and bridges laboratory-based physiology studies with real-world conditions. IMPORTANCEMetal-chelating chemicals are widely used across industries, including as preservatives in the food sector, but their full impact on microbial physiology is not well understood. We identified two genes, cciT and cciO, that function together to support Caulobacter crescentus iron balance when cells are exposed to the common synthetic chelator, EDTA. CciT is an outer membrane transporter and CciO is a dioxygenase-family protein that are mutually conserved in many bacteria, including several human pathogens, where mutations in cciT homologs are linked to clinical resistance to the siderophore antibiotic, cefiderocol. This study identifies a conserved genetic system that supports iron homeostasis during chelation stress and illuminates the iron acquisition versatility and stress resilience of Caulobacter in freshwater environments.

microbiology↗

bioRxiv2023

Untangling the Role of Pathobionts from Bacteroides Species in Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) arise from a convergence of underlying genetic susceptibility, environmental factors, and shifts in gut microbiota function and membership. Although the latter may trigger and contribute to IBD, there is little consensus on a specific causative pathogen. In this study, we demonstrate that commensal Bacteroides fragilis strains from ulcerative colitis (UC) patients before and during the development of ileal pouchitis engraft and promote colitis in specific pathogen free (SPF) IL-10 deficient (IL-10-/-) mice, but not in wild type SPF mice or when mono-associated in germ free mice. The colitis in IL-10-/- mice was also associated with significant alterations in commensal microbiota potentially important for maintaining intestinal and immune homeostasis. UC pouchitis B. fragilis also engrafts in DSS-induced colitis in WT SPF mice, indicating a fitness advantage under conditions of mucosal inflammation over other commensals in the gut microbiota. These findings show that gut inflammation promotes the expansion and fitness of UC-derived Bacteroides species that is associated with changes in the SPF gut microbiota and may be promote colitis in genetically susceptible hosts. ImportanceThis study supports the notion that human inflammatory bowel diseases arise from the emergence of indigenous pathobionts in genetically-prone subjects. Colitis-promoting pathobionts are well-suited to establish themselves in the host inflammatory environment and outcompete endogenous microbiota. Once engrafted, the pathobiont can further aggravate inflammation in a genetically-susceptible host. Such complex interplay among several factors creates a vicious pro-inflammatory cycle and promotes disease development. These findings are consistent with our previous clinical observation that B. fragilis, an otherwise low-abundance commensal species, expands prior to the development of UC pouchitis. We believe these findings are relevant to the pathogenesis of UC pouchitis and possibly human inflammatory bowel diseases in general, underscoring the role of commensal to pathobiont transitions, rather than classical pathogens, in promoting and exacerbating the onset of human IBD.

microbiology↗

bioRxiv2023

Microbially-catalyzed conjugation of GABA and tyramine to bile acids

Bile acids (BAs) are cholesterol-derived molecules that aid in digestion and nutrient absorption, regulate host metabolic processes, and influence physiology of the gut microbiota. Both the host and its microbiome contribute to enzymatic modifications that shape the chemical diversity of BAs in the gut. Several bacterial species have been reported to conjugate standard amino acids to BAs, but it was not known if bacteria conjugate BAs to other amine classes. Here, we show that Bacteroides fragilis strain P207, isolated from a bacterial bloom in the J-pouch of a patient with ulcerative colitis (UC) pouchitis, conjugates standard amino acids and the neuroactive amines {gamma}-aminobutyric acid (GABA) and tyramine to deoxycholic acid. We extended this analysis to other human gut isolates and identified species that are competent to conjugate GABA and tyramine to primary and secondary BAs, and further identified diverse BA-GABA and BA-tyramine amides in human stool. A longitudinal metabolomic analysis of J-pouch contents of the patient from whom B. fragilis P207 was isolated revealed highly reduced levels of secondary bile acids and a shifting BA amide profile before, during, and after onset of pouchitis, including temporal changes in several BA-GABA amides. Treatment of pouchitis with ciprofloxacin was associated with a marked reduction of nearly all BA amides in the J-pouch. Our study expands the known repertoire of conjugated bile acids produced by bacteria to include BA conjugates to GABA and tyramine and demonstrates that these molecules are present in the human gut. ImportanceBile acids (BAs) are modified in multiple ways by host enzymes and the microbiota to produce a chemically diverse set of molecules that assist in the digestive process and impact many physiological functions. This study reports the discovery of bacteria isolated from the gut of human patients that conjugate the neuroactive amines, GABA and tyramine, to BAs and demonstrates that BA-GABA and BA-tyramine amides are present in the human gut. GABA and tyramine are common metabolic products of the gut microbiota and potent neuroactive molecules, and their conjugation to BAs may influence receptor-mediated regulatory mechanisms of humans and their gut microbes.

microbiology↗

bioRxiv2023

XRE Transcription Factors Conserved in Caulobacter and {varphi}CbK Modulate Adhesin Development and Phage Production

Upon infection, transcriptional shifts in both a host bacterium and its invading phage determine host and viral fitness. The xenobiotic response element (XRE) family of transcription factors (TFs), which are commonly encoded by bacteria and phages, regulate diverse features of bacterial cell physiology and impact phage infection dynamics. Through a pangenome analysis of Caulobacter species isolated from soil and aquatic ecosystems, we uncovered an apparent radiation of a paralogous XRE TF gene cluster, several of which have established functions in the regulation of holdfast adhesin development and biofilm formation in C. crescentus. We further discovered related XRE TFs across the class Alphaproteobacteria and its phages, including the {varphi}CbK Caulophage, suggesting that members of this gene cluster impact host-phage interactions. Here we show that that a closely related group of XRE proteins, encoded by both C. crescentus and {varphi}CbK, can form heteromeric associations and control the transcription of a common gene set, influencing processes including holdfast development and the production of {varphi}CbK virions. The {varphi}CbK XRE paralog, tgrL, is highly expressed at the earliest stages of infection and can directly repress transcription of hfiA, a potent holdfast inhibitor, and gafYZ, a transcriptional activator of prophage-like gene transfer agents (GTAs) encoded on the C. crescentus chromosome. XRE proteins encoded from the C. crescentus chromosome also directly repress gafYZ transcription, revealing a functionally redundant set of host regulators that may protect against spurious production of GTA particles and inadvertent cell lysis. Deleting host XRE transcription factors reduced {varphi}CbK burst size, while overexpressing these genes or {varphi}CbK tgrL rescued this burst defect. We conclude that an XRE TF gene cluster, shared by C. crescentus and {varphi}CbK, plays an important role in adhesion regulation under phage-free conditions, and influences host-phage dynamics during infection.

microbiology↗

bioRxiv2023

The Caulobacter NtrB-NtrC two-component system bridges nitrogen assimilation and cell development

A suite of molecular sensory systems enables Caulobacter to control growth, development, and reproduction in re-sponse to levels of essential elements. The bacterial enhancer binding protein (bEBP) NtrC, and its cognate sensor histidine kinase NtrB, are key regulators of nitrogen assimilation in many bacteria, but their roles in Caulobacter metab-olism and development are not well defined. Notably, Caulobacter NtrC is an unconventional bEBP that lacks the {sigma}54- interacting loop commonly known as the GAFTGA motif. Here we show that deletion of C. crescentus ntrC slows cell growth in complex medium, and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glutamine synthetase (glnA) expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring transcription of the glnBA operon, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nutri-ent limitation. We further identified dozens of direct NtrC binding sites on the C. crescentus chromosome, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of binding sites align with those of the essential nucleoid associated protein, GapR, or the cell cycle regulator, MucR1. NtrC is therefore predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter. ImportanceBacteria balance cellular processes with the availability of nutrients in their environment. The NtrB-NtrC two-component signaling system is responsible for controlling nitrogen assimilation in many bacteria. We have characterized the effect of ntrB and ntrC deletion on Caulobacter growth and development and uncovered a role for spontaneous IS element transposition in the rescue of transcriptional and nutritional deficiencies caused by ntrC mutation. We further defined the regulon of Caulobacter NtrC, a bacterial enhancer binding protein, and demonstrate that it shares specific binding sites with essential proteins involved in cell cycle regulation and chromosome organization. Our work provides a com-prehensive view of transcriptional regulation mediated by a distinctive NtrC protein, establishing its connection to nitro-gen assimilation and developmental processes in Caulobacter.

microbiology↗

bioRxiv2023

Multi-omics analysis of a Bacteroides fragilis isolate from an ulcerative colitis patient defines genetic determinants of fitness in bile

Bacteroides fragilis comprises 1-5% of the gut microbiota in healthy humans but can expand to >50% of the population in ulcerative colitis (UC) patients experiencing inflammation. The mechanisms underlying such microbial blooms are poorly understood, but the gut of UC patients has physicochemical features that differ from healthy patients and likely impact microbial physiology. For example, levels of the secondary bile acid deoxycholate (DC) are highly reduced in the ileoanal J-pouch of UC colectomy patients. We isolated a B. fragilis strain from a UC patient with pouch inflammation (i.e. pouchitis) and developed it as a genetic model system to identify genes and pathways that are regulated by DC and that impact B. fragilis fitness in DC and crude bile. Treatment of B. fragilis with a physiologically relevant concentration of DC reduced cell growth and remodeled transcription of one-quarter of the genome. DC strongly induced expression of chaperones and select transcriptional regulators and efflux systems and downregulated protein synthesis genes. Using a barcoded collection of {approx}50,000 unique insertional mutants, we further defined B. fragilis genes that contribute to fitness in media containing DC or crude bile. Genes impacting cell envelope functions including cardiolipin synthesis, cell surface glycosylation, and systems implicated in sodium-dependent bioenergetics were major bile acid fitness factors. As expected, there was limited overlap between transcriptionally regulated genes and genes that impacted fitness in bile when disrupted. Our study provides a genome-scale view of a B. fragilis bile response and genetic determinants of its fitness in DC and crude bile. ImportanceThe Gram-negative bacterium, Bacteroides fragilis, is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation.

microbiology↗

bioRxiv2023

Cross regulation in a three-component cell envelope stress signaling system

A multi-layered structure known as the cell envelope separates the controlled interior of bacterial cells from a fluctuating physical and chemical environment. The transcription of genes that determine cell envelope structure and function is commonly regulated by two-component signaling systems (TCS), comprising a sensor histidine kinase and a cognate response regulator. To identify TCS genes that contribute to cell envelope function in the intracellular mammalian pathogen, Brucella ovis, we subjected a collection of non-essential TCS deletion mutants to compounds that disrupt cell membranes and the peptidoglycan cell wall. Our screen led to the discovery of three TCS proteins that coordinately function to confer resistance to cell envelope stressors and to support B. ovis replication in the intracellular niche. This tripartite regulatory system includes the known cell envelope regulator, CenR, and a previously uncharacterized TCS, EssR-EssS, which is widely conserved in Alphaproteobacteria. The CenR and EssR response regulators bind a shared set of sites on the B. ovis chromosomes to control transcription of an overlapping set of genes with cell envelope functions. CenR directly interacts with EssR and functions to stimulate phosphoryl transfer from the EssS kinase to EssR, while CenR and EssR control the cellular levels of each other via a post-transcriptional mechanism. Our data provide evidence for a new mode of TCS cross-regulation in which a non-cognate response regulator affects both the activity and protein levels of a cognate TCS protein pair. ImportanceAs intracellular pathogens, Brucella must contend with a variety of host-derived stressors when infecting a host cell. The inner membrane, cell wall, and outer membrane -- i.e. the cell envelope -- of Brucella provides a critical barrier to host assault. A conserved regulatory mechanism known as two-component signaling (TCS) commonly controls transcription of genes that determine the structure and biochemical composition of the cell envelope during stress. We report the identification of previously uncharacterized TCS genes that determine B. ovis fitness in the presence of cell envelope disruptors and within infected mammalian host cells. Our study reveals a new molecular mechanism of TCS-dependent gene regulation, and thereby advances fundamental understanding of transcriptional regulatory processes in bacteria.

microbiology↗

bioRxiv2022

A cryptic transcription factor regulates Caulobacter adhesin development

Alphaproteobacteria commonly produce an adhesin that is anchored to the exterior of the envelope at one cell pole. In Caulobacter crescentus this adhesin, known as the holdfast, facilitates attachment to solid surfaces and cell partitioning to air-liquid interfaces. An ensemble of two-component signal transduction (TCS) proteins controls C. crescentus holdfast biogenesis by indirectly regulating expression of HfiA, a potent inhibitor of holdfast synthesis. We performed a genetic selection to discover direct hfiA regulators that function downstream of the adhesion TCS system and identified rtrC, a hypothetical gene. rtrC transcription is directly activated by the adhesion TCS regulator, SpdR. Though its primary structure bears no resemblance to any defined protein family, RtrC binds and regulates dozens of sites on the C. crescentus chromosome via a pseudo-palindromic sequence. Among these binding sites is the hfiA promoter, where RtrC functions to directly repress transcription and thereby activate holdfast development. Either RtrC or SpdR can directly activate transcription of a second hfiA repressor, rtrB. Thus, environmental regulation of hfiA transcription by the adhesion TCS system is subject to control by an OR-gated type I coherent feedforward loop; these regulatory motifs are known to buffer gene expression against fluctuations in regulating signals. We have further assessed the functional role of rtrC in holdfast-dependent processes, including surface adherence to a cellulosic substrate and formation of pellicle biofilms at air-liquid interfaces. Strains harboring insertional mutations in rtrC have a diminished adhesion profile in a competitive cheesecloth binding assay and a reduced capacity to colonize pellicle biofilms in select media conditions. Our results add to an emerging understanding of the regulatory topology and molecular components of a complex bacterial cell adhesion control system. Author SummaryA complex structure known as the envelope separates the controlled interior of bacterial cells from the external environment. The envelope regulates molecular traffic in and out of the cell and mediates physical contact with the cells surroundings. Bacteria often anchor specialized polymers to the exterior of their envelopes, which enable attachment to surfaces and facilitate the development of multicellular communities known as biofilms. We have discovered that an uncharacterized hypothetical gene, present in common soil and aquatic bacteria, functions to control development of a surface adhesin known as the holdfast. This gene, which we have named rtrC, encodes a DNA-binding protein that regulates the expression of dozens of genes in Caulobacter. The expression of rtrC results in potent activation of holdfast biosynthesis, and loss of rtrC results in defects holdfast-dependent processes in Caulobacter including the ability to colonize biofilms at the surface of water. The results presented in this study illuminate the molecular function of previously hypothetical gene, and inform understanding of the molecular processes and pathways that control bacterial adhesion and biofilm development.

microbiology↗

bioRxiv2021

The ChvG-ChvI and NtrY-NtrX two-component systems coordinately regulate growth of Caulobacter crescentus

Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases directly phosphorylate their cognate response regulator to affect gene expression. Bacteria typically express multiple TCSs that are insulated from one another and regulate distinct physiological processes. There are certainly examples of cross-regulation between TCSs, but this phenomenon remains relatively unexplored. We have identified regulatory links between the ChvG-ChvI (ChvGI) and NtrY-NtrX (NtrYX) TCSs, which control important and often overlapping processes in -proteobacteria, including maintenance of the cell envelope. Deletion of chvG and chvI in Caulobacter crescentus limited growth in defined medium and a selection for genetic suppressors of this growth phenotype uncovered interactions among chvGI, ntrYX, and ntrZ, which encodes a previously uncharacterized periplasmic protein. Significant overlap in the experimentally-defined ChvI and NtrX transcriptional regulons provided support for the observed genetic connections between ntrYX and chvGI. Moreover, we present evidence that the growth defect of strains lacking chvGI is influenced by the phosphorylation state of NtrX and, to some extent, by levels of the TonB-dependent receptor ChvT. Measurements of NtrX phosphorylation in vivo indicated that NtrZ is an upstream regulator of NtrY, and that NtrY primarily functions as an NtrX phosphatase. We propose a model in which NtrZ functions in the periplasm to inhibit NtrY phosphatase activity; regulation of phosphorylated NtrX levels by NtrZ and NtrY provides a mechanism to modulate and balance expression of the NtrX and ChvI regulons under different growth conditions. ImportanceTwo-component signaling systems (TCSs) enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell envelope function in many -proteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. In addition, we identified NtrZ as a periplasmic inhibitor of NtrY phosphatase activity in vivo. Our work establishes C. crescentus as an excellent model to investigate multi-level regulatory connections between ChvGI and NtrYX in -proteobacteria.

microbiology↗

bioRxiv2020

Cysteine biosynthesis is a determinant of Brucella ovis stress survival and fitness in the intracellular niche

Brucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in axenic cultures during stationary phase. Studies of stationary phase may thus inform understanding of Brucella infection biology, yet the genes and pathways that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine, and we provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis at steps downstream of CysE. Deleting either cysE ({Delta}cysE) or both cysK1 and cysK2 ({Delta}cysK1 {Delta}cysK2) results in premature entry into stationary phase, reduced culture yield and sensitivity to exogenous hydrogen peroxide. These phenotypes can be chemically complemented by cysteine or glutathione. {Delta}cysE and {Delta}cysK1 {Delta}cysK2 strains have no defect in host cell entry in vitro but have significantly diminished intracellular fitness between 2 and 24 hours post infection. Our study has uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis, and demonstrates that cysteine anabolism is a determinant of peroxide stress survival and fitness in the intracellular niche.

microbiology↗

bioRxiv2020

Quantification of Brucella abortus population structure in a natural host

Cattle are natural hosts of the intracellular pathogen, Brucella abortus, which inflicts a significant burden on the health and reproduction of these important livestock. The primary routes of infection in field settings have been described, but it is not known how the bovine host shapes the structure of B. abortus populations during infection. We utilized a library of approximately 106 uniquely barcoded B. abortus strains to temporally and spatially quantify population structure at the strain level during colonization of cattle through a natural route of infection. Introducing 108 bacteria from this barcoded library to the conjunctival mucosa resulted in expected levels of local lymph node colonization at a one-week timepoint. We leveraged variance in strain abundance in the library to demonstrate that only 1 in 10,000 brucellae introduced at the site of infection reached the parotid lymph nodes. Thus, cattle restrict the overwhelming majority of B. abortus introduced via the ocular conjunctiva at this dose. Individual strains were spatially restricted within the host tissue, and the total B. abortus census was dominated by a small number of distinct strains in each lymph node. These results define a bottleneck that B. abortus must traverse to colonize local lymph nodes from the conjunctival mucosa. The data further support a model in which a small number of spatially isolated granulomas founded by unique strains are present one-week post infection. These experiments demonstrate the power of barcoded transposon tools to quantify infection bottlenecks and to define pathogen population structure in host tissues. Significance statementUnderstanding microbial population dynamics during infection has important implications for disease management, transmission and pathogen evolution. A quantitative analysis of microbial population structure requires the ability to track individual strains. We used a pool of individually barcoded strains to measure changes in Brucella abortus population structure during infection of bovine hosts via the ocular conjunctiva, a natural route of entry. Cattle exert a severe bottleneck on the bacterial population entering through the conjunctival mucosa such that individual cells have a 0.0001 probability of colonizing a local draining lymph node. The populations in lymph nodes, even on different sides of the same animal, are distinct and dominated by a small number of highly abundant, spatially distinct clones.

microbiology↗