Findings establish TodK as a modulator of developmental progression and demonstrate how quantitative phenotyping approaches can reveal biologically meaningful functions for orphan histidine kinases whose mutant phenotypes might otherwise appear subtle.
Abstract
Environmental bacteria have abundant signaling systems wired into complex gene regulatory networks to adapt to fluctuating conditions. In Myxococcus xanthus, starvation triggers a developmental program (specialized biofilm) that produces spore-filled multicellular fruiting bodies surrounded by a distinct quiescent state termed peripheral rods. Fruiting body structure as well as the proportion of cells following each fate can be tuned by a large repertoire of signaling proteins, including numerous orphan histidine kinases. Here, we focus on the histidine kinase TodK which was previously demonstrated to influence developmental progression. We find that loss of TodK produces distinct developmental phenotypes that vary with environmental conditions. To quantify these effects, we developed an image-analysis pipeline that measures aggregation and fruiting body patterning during development on nutrient-limited agar. These analyses revealed the todK mutant precociously aggregates particularly at the peripheries of the colony. Under submerged-culture conditions, initial production of aggregates was not accelerated but aggregates exhibited accelerated progression to mature fruiting bodies. Overexpression of active TodK completely blocked fruiting body formation. Molecular analyses demonstrated that TodK overproduction suppressed expression of core developmental regulators including FruA and CsgA (C-signal). Interestingly, protein accumulation of MrpC, necessary for expression of both FruA and the C-signal was not significantly perturbed suggesting TodK silences MrpC transcriptional activity. Together, these findings establish TodK as a modulator of developmental progression and demonstrate how quantitative phenotyping approaches can reveal biologically meaningful functions for orphan histidine kinases whose mutant phenotypes might otherwise appear subtle. Summary Statement Quantitative analysis of multicellular development reveals previously hidden functions of an orphan histidine kinase, highlighting the importance of robust phenotyping approaches for understanding bacterial signaling networks.
Myxococcus xanthus bacteria form aggregates when starved on solid surfaces and some cells differentiate into spores. Studies of mutants in monoculture have advanced knowledge of this multi-cellular developmental process, but our understanding of the genetic determinants is incomplete. To assess gene function genomewide, we generated a pool of barcoded transposon insertion mutants, subjected it to starvation, and separated developmental samples into non-aggregated cells, aggregated cells, and spores. We also subjected our pool to chemically-induced unicellular sporulation. Evaluation of changes in the abundance of mutants in samples allowed identification of 200 genes in which insertions reproducibly caused distinct patterns of depletion and/or accumulation over time. Many of these genes have well-established roles in development, validating our approach, while many others have not previously been associated with development. Genes involved in type IV pili (T4P)-dependent motility were more important than gliding motility genes for aggregation and sporulation in the mixed population. Although exopolysaccharide (EPS) synthesis genes are required for aggregation in monoculture, most were dispensable for aggregation in our pool, consistent with EPS sharing between cells, yet these genes were required cell-autonomously for efficient sporulation. Genes for positive regulators of EPS synthesis were important for aggregation as well as sporulation, suggesting functions beyond EPS production. Insertions in several novel genes impaired both starvation- and chemically-induced sporulation. Many genes increased the efficiency of starvation-induced sporulation. Some of these mutants, which we call “developmental winners”, are novel cheaters. Our results demonstrate the power of using the newly-created mutant library to elucidate M. xanthus biology. IMPORTANCE How cells coordinate their activities to build multicellular structures with differentiated cell types is a fundamental question in developmental biology. Starvation triggers thousands of M. xanthus cells to move coordinately and build mounds in which some cells differentiate into spores, while other cells lyse or persist as rods. We tracked a barcoded transposon mutant library through development with separation of sub-populations based on aggregation and sporulation fates. We discovered gene sets with distinct abundance profiles over time and across sub-populations. Sets contained both known and uncharacterized genes. For known genes, comparison of our results in a developmentally-competent mixture of mutants with published results for mutants in monoculture distinguished social from cell-autonomous functions. The novel genes provide numerous avenues toward deeper understanding of cellular interactions and differentiation. The mutant library offers a platform for further studies aimed at dissecting M. xanthus behaviors functionally, ecologically, and evolutionarily.
Sheenu Mittal, Saikat Mandal, Mark A. Farrugia et al.· bioRxiv· 0 citations
It is shown that the hyphae of the filamentous fungus Trichoderma guizhouense can differentiate into typical terminal and intercalary chlamydospores characterized by double-layered spherical or ellipsoidal cell walls with accumulated lipid bodies and nuclei, indicating the entry of cells into dormancy.
Yiting Hou, Yifan Li, Cheng Chen et al.· Applied and Environmental Mi...· 0 citations
ABSTRACT Quorum sensing (QS) enables microorganisms—including bacteria, eukaryotes, and viruses—to coordinate collective behaviors in response to population density. Despite their ecological and evolutionary significance, QS mechanisms in Archaea remain poorly characterized. The halophilic archaeon Haloferax volcanii provides a model for archaeal QS, transitioning from motile rods to non-motile disks in a density-dependent response to a secreted disk-forming signal (DFS). To identify components of the DFS regulatory network, we screened for spontaneous mutants that retained motility in DFS-containing soft-agar medium. One candidate, HVO_1357, encodes a predicted response regulator located adjacent to a histidine kinase (HVO_1356) and a second response regulator (HVO_1358), consistent with an extended two-component regulatory system (TCS). Based on our results, these genes encode quorum-sensing-associated regulators (Qar); therefore, we propose renaming them to qarA (HVO_1357), qarB (HVO_1356), and qarC (HVO_1358). Deletion of qarA enabled cells to swim on DFS-containing soft-agar plates and conferred hypermotility on standard soft-agar media; however, these phenotypes were not due to changes in motility-related parameters, but a reduced sensitivity to DFS for induction of the non-motile, disk-shaped state. In contrast, ΔqarB and ΔqarC strains were non-motile and exhibited premature disk formation during normal growth. Suppressor mutations restoring motility to ΔqarB and ΔqarC mapped exclusively to qarA, and epistasis analysis indicated QarA as the central regulator of this system. Phosphoablative variants of QarA, QarB, and QarC failed to complement their respective deletion strains, supporting QarABC as a TCS. Transcriptomic analyses revealed that qarA deletion leads to upregulation of genes involved in motility and rod-shape formation. Together, these findings reveal qarABC as a DFS-responsive regulatory module and represent the first TCS in archaea shown to control QS-dependent behavior. IMPORTANCE Archaea are ubiquitous and play key roles across diverse ecosystems—including human microbiomes—yet little is known about how they communicate with one another and with other organisms, or how these interactions shape their ecological impact. Such intercellular communication, including quorum sensing (QS), allows microorganisms to coordinate behaviors critical for survival, adaptation, and community organization. In this study, we identify the first archaeal two-component regulatory system that is involved in QS-dependent regulation, providing a foundation for understanding how organisms in this domain sense and respond to population cues. By revealing a previously unknown aspect of archaeal biology, this work represents an important step toward understanding how archaeal communication shapes both their physiology and their interactions within complex microbial communities. Archaea are ubiquitous and play key roles across diverse ecosystems—including human microbiomes—yet little is known about how they communicate with one another and with other organisms, or how these interactions shape their ecological impact. Such intercellular communication, including quorum sensing (QS), allows microorganisms to coordinate behaviors critical for survival, adaptation, and community organization. In this study, we identify the first archaeal two-component regulatory system that is involved in QS-dependent regulation, providing a foundation for understanding how organisms in this domain sense and respond to population cues. By revealing a previously unknown aspect of archaeal biology, this work represents an important step toward understanding how archaeal communication shapes both their physiology and their interactions within complex microbial communities.
Jacob A. Cote, Priyanka Chatterjee, Marco Garcia et al.· mBio· 1 citation
Phytopathogens encounter highly heterogeneous environments during infection. Carbon availability is a fundamental metabolic constraint that impacts bacterial virulence; however, the mechanism underlying the coordination of virulence traits based on metabolic cues remains unclear. We investigated the regulatory network of the soft-rot pathogen Pectobacterium carotovorum during Napa cabbage colonization, which generates tissue maceration with steep glucose and pectin gradients. Using GFP transcriptional fusions, we mapped the spatiotemporal expression of key regulatory (flhD, rsmA, and rsmB) and functional (fliC and pelA) virulence genes across necrotic lesions. As disease progressed, glucose and pectin accumulated at lesion centers before dissipating, establishing a distinct chemical zonation that mirrored virulence-gene profiles. Flagellar genes and rsmA showed consistent upregulation at lesion margins via the glucose-responsive cAMP-CRP system. In contrast, rsmB was upregulated at both centers and margins, whereas plant cell wall-degrading enzymes (PCWDEs) maintained relatively uniform expression. This spatial differentiation was driven by the concerted action of cAMP-CRP and pectin-responsive KdgR. cAMP-CRP disruption but not that of KdgR abolished the spatial organization of virulence gene expression and impaired lesion expansion, underscoring its central role in infection dynamics. Additionally, identification of previously unrecognized CRP-dependent targets reveals its expanded regulatory scope in Pectobacterium. These findings indicate a sophisticated spatiotemporal program where the interplay between cAMP-CRP and KdgR prioritizes motility at nutrient-depleted regions for outward expansion while sustaining PCWDE production across the lesion to maximize tissue maceration. This study highlights how carbon-responsive networks enable pathogens to exploit the complex chemical landscape of host tissues.
Yejin Park, Jae Hoon Lee· Microbiology Research· 0 citations
Bacterial toxin-antitoxin (TA) systems are classically viewed as stress-activated toxic switches. Specifically, ribonucleolytic toxins are thought to indiscriminately cleave RNA to halt cellular growth. We recently showed that the MazF toxin of Bacillus subtilis targets an unusually strict 6bp RNA cleavage sequence, but the implications of this stringent specificity were unknown. Here, we demonstrate that the MazEF system functions as a non-lethal post-transcriptional regulator in B. subtilis. Using a specialized single cell fluorescent reporter and transcriptome profiling, we show that MazF is uniformly activated across the population upon entry into the stationary phase, where it cleaves a narrow mRNA regulon to reshape gene expression. Rather than inhibiting growth, MazF activation tunes down the Spo0A stress response by repressing the mRNA level of its kinases. Reduced stress leads to an adaptive shortening of the lag phase upon nutrient replenishment. Furthermore, MazEF’s structural architecture, cleavage specificity, and impact on growth recovery are highly conserved across Gram-positive bacteria. Altogether, our findings redefine a paradigmatic toxin as a precision global mRNA stress regulator that primes cells for rapid regrowth. Graphical abstract
Regev Frenkel, Shira Omer, Tom Borenstein et al.· bioRxiv· 0 citations
Redox-based chemical warfare is a primary driver of microbial community assembly. Here, we show that the predatory bacterium Myxococcus xanthus employs a spatial division of labor between two inducible monofunctional catalases, mxKatB and mxKatE, to overcome prey-derived hydrogen peroxide (H₂O₂). Quantitative transcript analysis revealed distinct regulatory specificities: mxkatB was the dominant transcriptional responder to exogenous H₂O₂, whereas mxkatE was preferentially induced by UV irradiation. Biochemical analyses demonstrated strict compartmentalization of enzymatic activity. mxKatE functioned intracellularly, consistent with a role in mitigating endogenous genotoxic stress. In contrast, mxKatB, which harbors an N-terminal Sec-dependent signal peptide, was exclusively localized to the extracellular milieu. Targeted gene deletions corroborated these non-redundant physiological roles. ΔkatE mutant exhibited severe growth defects and heightened sensitivity to UV and H₂O₂ yet retained full predation proficiency. Conversely, ΔkatB mutant displayed unaltered vegetative fitness but were severely impaired in prey lysis due to oxidative inactivation of secreted bacteriolytic enzymes. Failure of cross-complementation confirmed that spatial localization, rather than catalytic capacity, dictates enzyme function. Our findings establish that M. xanthus deploys an extracellular catalase shield to protect its exoenzyme arsenal from prey-derived oxidants. This spatial specialization of antioxidant defenses represents a sophisticated strategy that directly determines the outcome of bacterial predation and shapes interspecies interactions within microbial communities. IMPORTANCE Predatory bacteria such as M. xanthus must withstand chemical defenses deployed by their prey. We show that M. xanthus uses a spatially specialized antioxidant system: an extracellular catalase (mxKatB) secreted to shield its lytic enzymes from prey-derived hydrogen peroxide, and an intracellular catalase (mxKatE) that handles endogenous oxidative stress. This division of labor reveals that bacterial antioxidant defenses can be compartmentalized to protect extracellular weaponry rather than the cell itself, adding a new dimension to how spatial organization of stress responses influences the outcome of microbial competition.
D. Sheng, Xuan-qi Zhang, Xin-yao Yan et al.· bioRxiv· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
