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Conjugated linoleic acid (CLA) possesses anticancer, anti-inflammatory, and antiobesity properties, making it a significant research focus. In this study, we identified TetR, a TetR/AcrR family transcriptional regulator encoded by AR1031 , as the transcriptional regulator of CLA synthesis in Lactiplantibacillus plantarum AR195. TetR binds to the promoter regions of the CLA synthesis genes, including the cla operon and cla-hy , thereby enhancing CLA biosynthesis. Knockout of tetR led to the downregulation of cla-hy by 68%, cla-dh by 86%, and cla-dc by 33%, resulting in reduced CLA yield. Further bioinformatic analysis and segmented electrophoretic mobility shift assay (EMSA) experiments revealed that TetR recognizes a conserved sequence (5'-TGTAGATTG-n 4 -CTTCA-3') and binds to multiple sites within the regulatory region of the cla operon and cla-hy , thus promoting CLA biosynthesis. Overexpression of tetR increased the CLA yield by 7%. These findings provide precise insights into the regulation of CLA biosynthesis and suggest strategies for enhancing CLA production.

Cra (catabolite repressor/activator) is a global transcription factor (TF) that plays a pleiotropic role in controlling the transcription of several genes involved in carbon utilization and energy metabolism. Multiple studies have investigated the regulatory mechanism of Cra and its rational use for metabolic regulation, but due to the complexity of its regulation, there remain challenges in the efficient use of Cra. Here, the structure, mechanism of action, and regulatory function of Cra in carbon and nitrogen flow are reviewed. In addition, this paper highlights the application of Cra in metabolic engineering, including the promotion of metabolite biosynthesis, the regulation of stress tolerance and virulence, the use of a Cra-based biosensor, and its coupling with other transcription factors. Finally, the prospects of Cra-related regulatory strategies are discussed. This review provides guidance for the rational design and construction of Cra-based metabolic regulation systems., Competing Interests: The authors declare no conflict of interest.

Catheter-associated urinary tract infections (CAUTIs) are a significant burden on healthcare systems, accounting for up to 40% of hospital-acquired infections globally. A prevalent CAUTI pathogen, Proteus mirabilis, is an understudied Gram-negative bacterium. One sequela of P. mirabilis CAUTI is the production of urinary stones, which complicates treatment and clearing of the infection. Stone formation is induced by the activity of urease, a nickel-metalloenzyme that is regulated by UreR in a urea-dependent manner. As urea is abundant in the urinary tract, urease genes are highly expressed during experimental UTI. We sought to leverage the urease promoter to create an expression system that would enable urea-inducible expression of genes during in vitro experiments as well as during experimental UTI. During preliminary studies, we observed unexpectedly high levels of basal expression of the urease promoter. This was somewhat dependent on the presence of regulator UreR. To further develop this expression system, we generated a series of reporter constructs to assess the impact of specific promoter elements on promoter activity in the presence and absence of urea. Elements of interest included known regulatory binding sites, alternative translational start sites, and single-nucleotide polymorphisms identified through comparative genomics. This work describes a suite of urea-inducible promoters, constructed during this study, that exhibit a variety of expression dynamics, providing a customizable platform for gene expression.IMPORTANCEUrea is an inexpensive molecule that can easily be supplied during in vitro experiments. A urea-inducible promoter would also be activated by environments where urea naturally occurs, such as in the urinary tract. Thus, the development of a urea-inducible system for selective gene expression is of great interest to the field of uropathogenesis as it would enable selective gene induction during experimental urinary tract infection. This expression system would also have important applications for recombinant protein production in biotech and manufacturing., Competing Interests: The authors declare no conflict of interest.

Type VII protein secretion systems play an important role in the survival and virulence of pathogens and in the competition among some microbes. Potential polymorphic toxin substrates of the type VII secretion system (T7SS) in Bacillus subtilis are important for competition in the context of biofilm communities. Within a biofilm, there is significant physiological heterogeneity as cells within the population take on differential cell fates. Which cells express and deploy the various T7SS substrates is still unknown. To identify which cells express at least one of the T7SS substrates, we investigated the yfj operon. The yfjABCDEF operon encodes at least one predicted T7SS substrate. Starting with an in silico analysis of the yfj operon promoter region, we identified potential regulatory sequences. Using a yfj promoter-reporter fusion, we then identified several regulators that impact expression of the operon, including a regulator of biofilm formation, DegU. In a degU deletion mutant, yfj expression is completely abolished. Mutation of predicted DegU binding sites also results in a significant reduction in yfj reporter levels. Further analysis of yfj regulation reveals that deletion of spo0A has the opposite effect of the degU deletion. Following the yfj reporter by microscopy of cells harvested from biofilms, we find that the yfj operon is expressed specifically in the subset of cells undergoing sporulation. Together, our results define cells entering sporulation as the subpopulation most likely to express products of the yfj operon in B. subtilis .IMPORTANCEDifferential expression of genes in a bacterial community allows for the division of labor among cells in the community. The toxin substrates of the type VII secretions system (T7SS) are known to be active in Bacillus subtilis biofilm communities. This work describes the expression of one of the T7SS-associated operons, the yfj operon, which encodes the YFJ toxin, in the sporulating subpopulation within a biofilm. The evidence that the YFJ toxin may be deployed specifically in cells at the early stages of sporulation provides a potential role for deployment of T7SS in community-associated activities, such as cannibalism., Competing Interests: The authors declare no conflict of interest.

The Gram-negative marine bacterium Vibrio anguillarum is able to cause vibriosis with hemorrhagic septicemia in many fish species, and iron acquisition is a critical step for virulence. Despite the fact that genes specific to certain processes of iron transport have been studied, the iron-regulated circuits of the V. anguillarum strains remain poorly understood. In this study, we showed that in V. anguillarum strain 775, iron could affect the expression of a number of critical metabolic pathways and virulence factors. The global iron uptake regulator Va Fur is the major actor to control these processes for the bacterium to respond to different iron conditions. A Va Fur binding motif was identified to distinguish directly and indirectly regulated targets. The absence of Va Fur resulted in the aberrant expression of most iron acquisition determinants under rich-iron conditions. A similar regulation pattern was also observed in the transcription of genes coding for the type VI secretion system. The expression of peroxidase genes is positively controlled by Va Fur to prevent iron toxicity, and the deletion of Vafur caused impaired growth in the presence of iron and H 2 O 2 . Va Fur also upregulates some virulence factors under limited-iron conditions, including metalloprotease EmpA and motility, which are likely critical for the high virulence of V. anguillarum 775. The deletion of Va Fur led to reduced swimming motility and decreased extracellular protease activity under limited-iron conditions, thereby leading to attenuated pathogenicity. Our study provides more evidence to better understand the Va Fur regulon and its role in the pathogenesis of V. anguillarum .IMPORTANCEVibriosis, the most common disease caused by marine bacteria belonging to the genus Vibrio , leads to massive mortality of economical aquatic organisms in Asia. Iron is one of the most important trace elements, and its acquisition is a critical battle occurring between the host and the pathogen. However, excess iron is harmful to cells, so iron utilization needs to be strictly controlled to adapt to different conditions. This process is mediated by the global iron uptake regulator Fur, which acts as a repressor when iron is replete. On the other hand, free iron in the host is limited, so the reduced virulence of the Δ fur mutant should not be directly caused by abnormally regulated iron uptake. The significance of this work lies in uncovering the mechanism by which the deletion of Fur causes reduced virulence in Vibrio anguillarum and identifying the critical virulence factors that function under limited-iron conditions., Competing Interests: The authors declare no conflict of interest.

Background: Plesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors., Results: In this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides' capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides., Conclusions: Overall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Shigella is a foodborne enteropathogenic bacteria that causes severe bacillary dysentery in humans. Shigella primarily colonizes the human colon and causes disease via invasion of colon epithelial cells. However, the signal regulatory mechanisms associated with its colonization and pathogenesis in the colon remain poorly defined. Here, we report a leucine-mediated regulatory mechanism that promotes Shigella virulence gene expression and invasion of colon epithelial cells. Shigella in response to leucine, which is highly abundant in the colon, via the leucine-responsive regulator Lrp and the binding of Lrp with leucine induces the expression of a newly identified small RNA SsrV. SsrV then activates the expression of virF and downstream invasion-related virulence genes by increasing the protein level of the LysR-type transcription regulator LrhA, therefore enabling Shigella invasion of colon epithelial cells. Shigella lacking ssrV displays impaired invasion ability. Collectively, these findings suggest that Shigella employs a leucine-responsive environmental activation mechanism to establish colonization and pathogenicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5'-TAGTTGA-C-TGGTCTA-3'. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Iron (Fe) is a trace nutrient required by nearly all organisms. As a result of the demand for Fe and the toxicity of non-chelated cytosolic ionic Fe, regulatory systems have evolved to tightly balance Fe acquisition and usage while limiting overload. In most bacteria, including the mammalian pathogen Staphylococcus aureus , the ferric uptake regulator (Fur) is the primary transcriptional regulator controlling the transcription of genes that code for Fe uptake and utilization proteins. Fpa (formerly YlaN) was demonstrated to be essential in Bacillus subtilis unless excess Fe is added to the growth medium, suggesting a role in Fe homeostasis. Here, we demonstrate that Fpa is essential in S. aureus upon Fe deprivation. Null fur alleles bypassed the essentiality of Fpa. The absence of Fpa abolished the derepression of Fur-regulated genes during Fe limitation. Bioinformatic analyses suggest that fpa was recruited to Gram-positive bacteria and, once acquired, was maintained in the genome as it co-evolved with Fur. Consistent with a role for Fpa in alleviating Fur-dependent repression, Fpa and Fur interacted in vivo , and Fpa decreased the DNA-binding ability of Fur in vitro . Fpa bound Fe(II) in vitro using oxygen or nitrogen ligands with an association constant that is consistent with a physiological role in Fe homeostasis. These findings have led to a model wherein Fpa is an Fe(II) binding protein that influences Fur-dependent regulation through direct interaction.IMPORTANCEIron (Fe) is an essential nutrient for nearly all organisms. If Fe homeostasis is not maintained, Fe may accumulate in the cytosol, which can be toxic. Questions remain about how cells efficiently balance Fe uptake and usage to prevent overload. Iron uptake and proper metalation of proteins are essential processes in the mammalian bacterial pathogen Staphylococcus aureus . Understanding the gene products involved in the genetic regulation of Fe uptake and usage and the physiological adaptations that S. aureus uses to survive in Fe-depleted conditions provides insight into pathogenesis. Herein, we demonstrate that the DNA-binding activity of the ferric uptake regulator transcriptional repressor is alleviated under Fe limitation, but uniquely, in S. aureus , alleviation requires the presence of Fpa., Competing Interests: The authors declare no conflict of interest.

The human pathogen Pseudomonas aeruginosa ( Pa ) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca 2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca 2+ -binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca 2+ -regulated virulence in P. aeruginosa . Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca 2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca 2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca 2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca 2+ and Fe, and this regulation required a Ca 2+ -dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca 2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca 2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to the host.IMPORTANCE Pseudomonas aeruginosa ( Pa ) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca 2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca 2+ sensor, EfhP, is required for at least 1/3 of the Ca 2+ response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of efhP itself is regulated by Ca 2+ and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca 2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca 2+ and associated regulatory mechanisms will serve in the development of future therapeutics targeting Pa 's dangerous infections., Competing Interests: The authors declare no conflict of interest.

Quorum sensing (QS) is a cell-to-cell communication process that enables bacteria to coordinate group behaviors. In Vibrio cholerae colonies, a program of spatial-temporal cell death is among the QS-controlled traits. Cell death occurs in two phases, first along the colony rim, and subsequently, at the colony center. Both cell death phases are driven by the type 6 secretion system (T6SS). Here, we show that HapR, the master QS regulator, does not control t6ss gene expression nor T6SS-mediated killing activity. Nonetheless, a Δ hapR strain displays no cell death at the colony rim. RNA-Sequencing (RNA-Seq) analyses reveal that HapR activates expression of an operon containing four genes of unknown function, vca0646-0649. Epistasis and overexpression studies show that two of the genes, vca0646 and vca0647 , are required to drive cell death in both a Δ hapR and a Δ hapR Δ t6ss strain. Thus, vca0646 - 0649 are regulated by HapR but act independently of the T6SS machinery to cause cell death, suggesting that a second, parallel pathway to cell death exists in V. cholerae ., Competing Interests: Competing interests statement:The authors declare no competing interest.

The opportunistic pathogen Pseudomonas aeruginosa infects the airways of people with cystic fibrosis (CF) and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen., (Published by Oxford University Press on behalf of Nucleic Acids Research 2024.)

Streptomycetes are bacteria of significant biotechnological interest due to their production of bioactive specialised metabolites used in medicine and agriculture. In these bacteria, specialised metabolism and morphological differentiation are linked and typically repressed under high phosphate conditions. This study characterises a DeoR-like transcriptional regulator, SCO1897, in Streptomyces coelicolor, whose expression increases during sporulation. Disruption of sco1897 results in reduced biosynthesis of specialised metabolites, delayed sporulation, higher spore phosphate content, and impaired germination. Transcriptomic analysis revealed 1420 genes differentially expressed in the sco1897 mutant compared to the S. coelicolor wild-type strain. The sco1897 gene is located upstream and transcribed in the same direction as six genes, including sco1898-1900 encoding sub-units of an ABC transporter annotated as involved in carbohydrate transport. SCO1897 negatively regulates its own expression, that of the sco1898-1900 ABC transporter, and sco4142, encoding the PstS phosphate-binding protein. The overexpression of sco1898-1900 in the S. coelicolor wild-type strain leads to a significant increase in intracellular spore phosphate levels, similar to those observed in the sco1897 mutant. These findings suggest a complex regulatory network involving the sco1897-sco1900 region. Hypotheses are proposed to explain the various phenotypes of the sco1897 mutant and the complex regulation of the genes of the sco1897-sco1900 region., (© 2024. The Author(s).)

Staphylococcus aureus can cause outbreaks and becomes multi-drug resistant through gene mutations and acquiring resistance genes. However, why S. aureus easily adapts to hospital environments, promoting resistance and recurrent infections, remains unknown. Here we show that a specific S. aureus lineage evolved from a clone that expresses the accessory gene regulator (Agr) system to subclones that reversibly suppressed Agr and caused an outbreak in the hospital setting. S. aureus with flexible Agr regulation shows increased ability to acquire antibiotic-resistant plasmids, escape host immunity, and colonize mice. Bacteria with flexible Agr regulation shows altered cytosine genomic methylation, including the decreased 5mC methylation in transcriptional regulator genes (pcrA and rpsD), compared to strains with normal Agr expression patterns. In this work, we discover how altered genomic methylation promotes flexible Agr regulation which is associated with persistent pathogen colonization in the hospital environment., (© 2024. The Author(s).)

NorR, as a single-target regulator, has been demonstrated to be involved in NO detoxification in bacteria under anaerobic conditions. Here, the norR gene was identified and deleted in the genome of Vibrio alginolyticus. The results showed that deletion of norR in Vibrio alginolyticus led to lower swarming motility and more biofilm formation on aerobic condition. Moreover, we proved that NorR from E. coli had a similar function in controlling motility. NorR overexpression led to increased resistance to oxidative stress and tetracycline. We also observed a reduced ability of the NorR-overexpressing strain to adapt to iron limitation condition. Transcriptome analysis showed that the genes responsible for bacterial motility and biofilm formation were affected by NorR. The expressions of several sigma factors (RpoS, RpoN, and RpoH) and response regulators (LuxR and MarR) were also controlled by NorR. Furthermore, Chip-qPCR showed that there is a direct binding between NorR and the promoter of rpoS. Based on these results, NorR appears to be a central regulator involved in biofilm formation and swarming motility in Vibrio alginolyticus., (© 2024. The Author(s).)

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phospho t ransferase s ystem (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes , has an EIIB Gat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes , harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification., Competing Interests: The authors declare no conflict of interest.

Bordetella bronchiseptica is a pathogenic bacterium that causes respiratory infections in mammals. Adhesins, toxins, and secretion systems necessary for infection are regulated by the two-component system BvgAS. When the BvgAS system is inactive, there is no transcription of virulence-activated genes, and virulence-repressed genes ( vrg ) are expressed. The regulation of some vrgs in B. bronchiseptica is dependent upon the virulence-activated gene bvgR . Although having a regulatory role, no DNA-binding domain is described for BvgR. Instead, it contains an EAL domain, usually found in cyclic-di-GMP (c-di-GMP)-specific phosphodiesterases. c-di-GMP is a bacterial second messenger that regulates multiple phenotypes in bacteria, including B. bronchiseptica . The current study aimed to deepen our knowledge about BvgR. We employed RNA-seq analysis to define the BvgR regulon, and then we investigated the phenotypes in which BvgR regulation might be involved such as biofilm formation, cytotoxicity, and virulence. Our result revealed that BvgR inhibits biofilm formation and flagellin expression in virulent phase. Although BvgR has long been considered a repressor protein, our results show that it also upregulates almost 100 genes. This regulation is likely indirect, as BvgR lacks a DNA-binding domain. Notably, among the upregulated genes, we identified 15 associated with the type three secretion system. Consistent with these findings, a B. bronchiseptica strain deficient in bvgR was less cytotoxic than the wild-type strain, elicited a milder immune response, and was less able to persist in the lower respiratory tract of mice.IMPORTANCE Bordetella bronchiseptica is a harmful bacterium responsible for respiratory infections in mammals. Its ability to cause disease is tightly regulated by a system called BvgAS. In this study, we focused on understanding the role of a specific gene called bvgR in regulating B. bronchiseptica 's virulence factors. Our findings revealed that BvgR, previously thought to primarily repress gene expression, actually plays a complex role in both activating and inhibiting various genes involved in bacterial virulence. This newfound understanding sheds light on the intricate mechanisms underlying B. bronchiseptica 's ability to cause infections, providing valuable insights for developing strategies to combat these infections in humans and animals., Competing Interests: The authors declare no conflict of interest.

For bacteria, an intricate coordination between sensing and regulating iron levels and managing oxidative stress is required as their levels are tightly interlinked. While various oxidative stress and heme-based redox sensors have been reported for both pathogenic and non-pathogenic bacteria, the mechanisms governing the modulation of intracellular iron levels in response to changes in redox status remain unclear. In this study, a gene-inactivated strain of mycobacterial sensor kinase PdtaS showed dysregulated expression of genes associated with iron metabolism, including Fe-S clusters, NADH dehydrogenases, and iron uptake. The strain showed poor growth in nutrient-limiting conditions, a defect rescuable by heme but not by Fe 3+ supplementation. This observation was associated with the PAS domain of the PdtaS sensor kinase. Biochemical and biophysical experiments established heme-binding to the PAS domain and its inhibitory effect on PdtaS auto-kinase activity, suggesting that the absence of heme induces activation of this sensor kinase. Interestingly, despite having an endogenous heme biosynthetic pathway or even external heme supplementation, the ∆ pdtaS mutant exhibited persistent low intracellular iron levels concomitant with elevated oxidative stress. Antioxidant supplementation mitigated growth defects, emphasizing the link between oxidative stress, intracellular iron levels, and PdtaS activity. RNA-IP identified key targets associated with redox homeostasis and iron metabolism as targets of the PdtaR response regulator. The study proposes a novel role for the PdtaS-PdtaR TCS in sensing heme, regulation of intracellular iron levels, and redox balance.IMPORTANCEThe research article investigates the intricate interplay between bacteria's ability to take and utilize iron without inducing excess iron's toxic effects, including oxidative stress. The study shows that bacteria achieve this by sensing intracellular iron available as heme through a sensory protein PdtaS, which turns off when heme is in excess and prevents iron uptake and iron efflux. The process shields bacteria from generating Fe-dependent free radicals and allows it to maintain viability. The absence of sensor kinase abrogates all these processes, increasing bacteria susceptibility to ROS and thereby slowing growth. This feature of the sensor kinase PdtaS makes it an attractive co-therapeutic target for tuberculosis therapy, where its inhibition will prevent iron uptake, even in the presence of low iron, thereby halting bacterial proliferation., Competing Interests: The authors declare no conflict of interest.

Background: Limited research has been conducted on energy fluctuation during the transition state, despite the critical role of energy supply in microbial physiological metabolism., Results: This study aimed to investigate the regulatory function of transition state transcription factor AbrB on energy metabolism in Bacillus licheniformis WX-02. Firstly, the deletion of abrB was found to prolong the cell generation time, significantly reducing the intercellular ATP concentration and NADH/NAD + ratio at the early stage. Subsequently, various target genes and transcription factors regulated by AbrB were identified through in vitro verification assays. Specifically, AbrB was shown to modulate energy metabolism by directly regulating the expression of genes pyk and pgk in substrate-level phosphorylation, as well as genes narK and narGHIJ associated with nitrate respiration. In terms of oxidative phosphorylation, AbrB not only directly regulated ATP generation genes, including cyd, atpB, hmp, ndh, qoxA and sdhC, but also influenced the expression of NAD-dependent enzymes and intracellular NADH/NAD + ratio. Additionally, AbrB positively affected the expression of transcription factors CcpN, Fnr, Rex, and ResD involved in energy supply, while negatively affected the regulator CcpA. Overall, this study found that AbrB positively regulates both substrate-level phosphorylation and oxidative phosphorylation, while negatively regulating nitrate respiration., Conclusions: This study proposes a comprehensive regulatory network of AbrB on energy metabolism in Bacillus, expanding the understanding of regulatory mechanisms of AbrB and elucidating energy fluctuations during the transition state., (© 2024. The Author(s).)

Fumarate and nitrate reduction regulatory protein (Fnr)-a global transcriptional regulator-can directly or indirectly regulate many genes in different metabolic pathways at the top of the bacterial transcription regulation network. The present study explored the regulatory mechanism of Fnr-mediated nitrite degradation in Lactobacillus plantarum WU14 through gene transcription and expression analysis of oxygen sensing and nir operon expression regulation by Fnr. The interaction and the mechanism of transcriptional regulation between Fnr and GlnR were also examined under nitrite stress. After Fnr and GlnR purification by glutathione S-transferase tags, they were successfully expressed in Escherichia coli by constructing an expression vector. The results of electrophoresis mobility shift assay and qRT-PCR indicated that Fnr specifically bound to the PglnR and Pnir promoters and regulated the expression of nitrite reductase (Nir) and GlnR. After 6-12 h of culture, the expressions of fnr and nir under anaerobic conditions were higher than under aerobic conditions; the expression of these two genes increased with sodium nitrite (NaNO 2 ) addition during aerobic culture. Overall, the present study indicated that Fnr not only directly participated in the expression of Nir and GlnR but also indirectly regulated the expression of Nir through GlnR regulation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The collective behavior of bacteria is regulated by quorum sensing (QS). Autoinducer-2 (AI-2) is a common QS signal that regulates the behavior of both Gram-positive and Gram-negative bacteria. Despite the plethora of processes described to be influenced by AI-2 in diverse Gram-negative bacteria, the AI-2-regulated processes in Bacilli are relatively unexplored. Here, we describe a novel function for AI-2 in Bacillus velezensis SQR9 related to the sporulation. AI-2 inhibited the initiation of sporulation through the phosphatase RapC and the DNA binding regulator ComA. Using biochemistry experiments, we demonstrated that AI-2 interacts with RapC to stimulate its binding to ComA, which leads to an inactive ComA and subsequently a sporulation inhibition. The AI-2 molecule could be shared across species for inhibiting Bacillus sporulation and it also plays the same role in different soil conditions. Our study revealed a novel function and regulatory mechanism of AI-2 in inhibiting sporulation in Bacilli., (© 2024. The Author(s).)

Listeria monocytogenes is a food-borne pathogen that must adapt to several environments both inside and outside the host. One such environment is the microaerophilic conditions encountered in the host intestine proximal to the mucosal surface. The aim of this study was to investigate the expression of the PrfA regulon in response to microaerophilic growth conditions in the presence of either glucose or glycerol as a carbon source using four transcriptional (P hly , P actA , P /prfA and P /plcA ) gene fusions. Further, RNAseq analysis was used to identify global changes in gene expression during growth in microaerophilic conditions. Following microaerophilic growth, there was a PrfA-dependent increase in transcription from the P hly , P actA and P /plcA promoters, indicating that microaerophilic growth induces the PrfA regulon regardless of the carbon source with increased expression of the PrfA, LLO and ActA proteins. A sigB mutation had no effect on the induction of the PrfA regulon under microaerophilic conditions when glucose was used as a carbon source. In contrast, when glycerol was the carbon source, a sigB mutation increased expression from the P hly and P actA promoters regardless of the level of oxygen. The RNAseq analysis showed that 273 genes were specifically regulated by microaerophilic conditions either up or down including the PrfA regulon virulence factors. Overall, these data indicated that L. monocytogenes PrfA regulon is highly responsive to the low-oxygen conditions likely to be encountered in the small intestine and that SigB has an input into the regulation of the PrfA regulon when glycerol is the sole carbon source.

Aerobic respiration is the key driver of Vibrio cholerae proliferation and infection. Our previous transcriptome results suggested that degS knockout downregulates a few genes involved in NADH and ATP synthesis in the aerobic respiratory pathway. In this study, non-targeted metabolomics results showed that the differential metabolites affected by degS knockout were associated with aerobic respiration. Further results suggested that the key products of aerobic respiration, NADH and ATP, were reduced upon degS deletion and were not dependent on the classical σ E pathway. The two-component system response factor aerobic respiration control A (ArcA) is involved in regulating NADH and ATP levels. qRT-PCR demonstrated that DegS negatively regulates the transcription of the arcA gene, which negatively regulates the expression of isocitrate dehydrogenase (ICDH), a key rate-limiting enzyme of the tricarboxylic acid cycle. NADH and ATP levels were partially restored with the knockout of the arcA gene in the ΔdegS strain, while levels were partially restored with overexpression of ICDH in the ΔdegS strain. In a growth experiment, compared to the ΔdegS strain, the growth rates of ΔdegSΔarcA and ΔdegS -overexpressed icdh strains ( ΔdegS+icdh ) were partially restored during the logarithmic growth period. Colonization of the intestines of suckling mice showed a significant reduction in the colonizing ability of the ΔdegS strain, similar colonizing ability of the ΔdegS::degS strain and the wild-type strain, and a partial recovery of the colonizing ability of the ΔdegS + icdh strain. Overall, these findings suggest that the DegS protease regulates the expression of ICDH through ArcA, thereby affecting the NADH and ATP levels of V. cholerae and its growth and intestinal colonization ability., Competing Interests: The authors declare that they have no competing financial interests or personal relationships that influenced the work reported in this paper., (Copyright © 2024 Zhao, Huang, Li, Ren, Hu, Yuan, Wang, Hu, Huang and Min.)

The RaxX sulfopeptide, secreted via a type Ι secretion system, is crucial for activating XA21-mediated innate immunity in resistant rice lines bearing the XA21 receptor kinase. Certain pathogenicity-associated regulators that control the expression of the raxSTAB-raxX gene cluster have been functionally characterized, but the comprehensive regulatory cascade of RaxSTAB and RaxX in Xanthomonas oryzae pv. oryzae (Xoo) remains incompletely understood. Our investigation revealed that pathogenicity-associated regulators, including HrpG, HrpX, VemR, PhoR, and Clp, form a regulatory cascade governing the expression of the raxSTAB-raxX gene cluster. HrpG regulates the raxSTAB-raxX gene cluster transcription through the key regulator HrpX. VemR also participates in the transcription of the raxSTAB-raxX. The histidine kinase PhoR positively modulates raxSTAB-raxX expression, while the global regulator Clp directly binds the raxX promoter region to promote its transcription. These findings shed light on the intricate regulatory cascade of rax-related genes in Xoo, emphasizing the complex roles of pathogenicity-associated regulators within the pathogenic regulatory system., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Cellular levels of the essential micronutrient manganese (Mn) need to be carefully balanced within narrow borders. In cyanobacteria, a sufficient Mn supply is critical for ensuring the function of the oxygen-evolving complex as the central part of the photosynthetic machinery. However, Mn accumulation is fatal for the cells. The reason for the observed cytotoxicity is unclear. To understand the causality behind Mn toxicity in cyanobacteria, we investigated the impact of excess Mn on physiology and global gene expression in the model organism Synechocystis sp. PCC 6803. We compared the response of the WT and the knock-out mutant in the Mn e x porter (Mnx), ∆ mnx , which is disabled in the export of surplus Mn and thus functions as a model for toxic Mn overaccumulation. While growth and pigment accumulation in ∆ mnx were severely impaired 24 h after the addition of tenfold Mn, the WT was not affected and thus mounted an adequate transcriptional response. RNA-seq data analysis revealed that the Mn stress transcriptomes partly resembled an iron limitation transcriptome. However, the expression of iron limitation signature genes isiABDC was not affected by the Mn treatment, indicating that Mn excess is not accompanied by iron limitation in Synechocystis . We suggest that the ferric uptake regulator, Fur, gets partially mismetallated under Mn excess conditions and thus interferes with an iron-dependent transcriptional response. To encounter mismetallation and other Mn-dependent problems on a protein level, the cells invest in transcripts of ribosomes, proteases and chaperones. In the case of the ∆ mnx mutant, the consequences of the disability to export excess Mn from the cytosol manifest in additionally impaired energy metabolism and oxidative stress transcriptomes with a fatal outcome. This study emphasizes the central importance of Mn homeostasis and the transporter Mnx's role in restoring and holding it.

Vibrio cholerae causes cholera, an important cause of death worldwide. A fuller understanding of how virulence is regulated offers the potential for developing virulence inhibitors, regarded as efficient therapeutic alternatives for cholera treatment. Here we show using competitive infections of wild-type and mutant bacteria that the regulator of chitosan utilization, ChsR, increases V. cholerae virulence in vivo. Mechanistically, RNA sequencing, chromatin immunoprecipitation with sequencing and molecular biology approaches revealed that ChsR directly upregulated the expression of the virulence regulator, TcpP, which promoted expression of the cholera toxin and the toxin co-regulated pilus, in response to low O 2 levels in the small intestine. We also found that chitosan degradation products inhibit the ChsR-tcpP promoter interaction. Consistently, administration of chitosan oligosaccharide, particularly when delivered via sodium alginate microsphere carriers, reduced V. cholerae intestinal colonization and disease severity in mice by blocking the chsR-mediated pathway. These data reveal the potential of chitosan oligosaccharide as supplemental therapy for cholera treatment and prevention., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Gallibacterium anatis is a member of the Pasteurellaceae family and is an opportunistic pathogen that causes gallibacteriosis in chickens. Stress plays a relevant role in promoting the development of pathogenicity in G. anatis. Epinephrine (E) and norepinephrine (NE) are relevant to stress; however, their effects on G. anatis have not been elucidated. In this work, we evaluated the effects of E and NE on the growth, biofilm formation, expression of adhesins, and proteases of two G. anatis strains, namely, the hemolytic 12656-12 and the nonhemolytic F149 T biovars. E (10 μM/mL) and NE (30 and 50 μM/mL) increased the growth of G. anatis 12656-12 by 20 % and 25 %, respectively. E did not affect the growth of F149 T , whereas 40 μM/mL NE decreased bacterial growth by 25 %. E and NE at a dose of 30-50 μM/mL upregulated five fibrinogen adhesins in the 12565-12 strain, whereas no effect was observed in the F149 T strain. NE increased proteolytic activity in both strains, whereas E diminished proteolytic activity in the 12656-12 strain. E and NE reduced biofilm formation (30 %) and increased Congo red binding (15 %) in both strains. QseBC is the E and NE two-component detection system most common in bacteria. The qseC gene, which is the E and NE receptor in bacteria, was identified in the genomic DNA of the 12565-12 and F149 T G. anatis strains via PCR amplification. Our results suggest that QseC can detect host changes in E and NE concentrations and that catecholamines can modulate the expression of several virulence factors in G. anatis., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Type III secretion system (T3SS) is an important virulence system in Gram-negative bacteria. In this investigation, different environmental conditions that regulate the expression of T3SS genes in Yersinia ruckeri were investigated aimed at obtaining a better understanding about its modulation after various environmental challenges. Four isolates of Y. ruckeri CSF007-82, ATCC29473, A7959-11 and YRNC10 were cultivated under the diverse in vitro challenges iron depletion, high salt, low pH and in the presence of fish serum or in the fish cell culture (Chinook Salmon Embryo - CHSE). The transcriptional modulation of the chromosomal genes ysaV, ysaC, ysaJ and prgH of ysa were investigated using quantitative real-time PCR. The expression of prgH, ysaV, ysaC and ysaJ was differentially expressed in all four strains under evaluation. The highest gene expression levels were observed for Y. ruckeri YRNC10 AN after addition of 0.3 M NaCl in Luria Bertani broth. The results obtained from this study provide initial insights into T3SS responses in Y. ruckeri, which pave the way for further studies aimed at expanding our knowledge on the functional roles of the T3SS genes in Y. ruckeri., (© 2024 The Author(s). Journal of Fish Diseases published by John Wiley & Sons Ltd.)

The alternative sigma factor RpoS functions as a regulator of stress and virulence response in numerous bacterial species. Vibrio mimicus is a critical opportunistic pathogen causing huge losses to aquaculture. However, the exact role of RpoS in V. mimicus remains unclear. In this study, rpoS deletion mutant of V. mimicus was constructed through allelic exchange and the phenotypic and transcriptional changes were investigated to determine the function of RpoS. The abilities of growth, motility, biofilm production, hemolytic activity and pathogenicity were significantly impaired in ΔrpoS strain. Stationary-phase cells of ΔrpoS strain showed lower tolerance to H 2 O 2 , heat, ethanol, and starvation stress than the wild-type strain. Transcriptome analyses revealed the involvement of rpoS in various cellular processes, notably bacterial-type flagellum synthesis and assembly, membrane synthesis and assembly and response to various stimuli. Phenotypic and RNA-seq analysis revealed that RpoS is required for biofilm formation, stress resistance, and pathogenicity in V. mimicus. Furthermore, β-galactosidase activity showed that rpoS is essential for optimal transcription of the flgK, fliA, cheA, mcpH mRNA. These results offer significant insight into the function and regulatory network of rpoS/RpoS, thereby improving our understanding and facilitating selection of molecular targets for future prevention strategies against V. mimicus., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Small RNA (sRNA) in bacteria serve as the key messengers in regulating genes associated with quorum sensing controlled bacterial virulence. This study was aimed to unveil the regulatory role of sRNA PA0730.1 on the expression of various traits of Pseudomonas aeruginosa linked to pathogenicity, with special emphasis on the growth, colony morphology, cell motility, biofilm formation, and the expression of diverse virulence factors. PA0730.1 sRNA was found to be upregulated both during planktonic stationary growth phase and at biofilm state of P. aeruginosa PAO1. PA0730.1 deleted strain showed significant growth retardation with increased doubling time. Overexpression of PA0730.1 led to enhanced motility and biofilm formation, while the ∆PA0730.1 strain displayed significant inhibition in motility and biofilm formation. Furthermore, PA0730.1 was found to regulate the synthesis of selected virulence factors of P. aeruginosa. These observations in PA0730.1 + and ∆PA0730.1 were found to be correlated with the PA0730.1-mediated repression of transcription regulators, mucA and rpoS, both at transcriptional and translational levels. The results suggest that PA0730.1 sRNA might be a promising target for developing new drug to counter P. aeruginosa pathogenesis, and could also help in RNA oligonucleotide based therapeutic research for formulating a novel therapeutant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phenolic compounds are commonly found in anoxic environments, where they serve as both carbon and energy sources for certain anaerobic bacteria. The anaerobic breakdown of m-cresol, catechol, and certain lignin-derived compounds yields the central intermediate 3-hydroxybenzoate/3-hydroxybenzoyl-CoA. In this study, we have characterized the transcription and regulation of the hbd genes responsible for the anaerobic degradation of 3-hydroxybenzoate in the β-proteobacterium Aromatoleum sp. CIB. The hbd cluster is organized in three catabolic operons and a regulatory hbdR gene that encodes a dimeric transcriptional regulator belonging to the TetR family. HbdR suppresses the activity of the three catabolic promoters (P hbdN , P hbdE and P hbdH ) by binding to a conserved palindromic operator box (ATGAATGAN 4 TCATTCAT). 3-Hydroxybenzoyl-CoA, the initial intermediate of the 3-hydroxybenzoate degradation pathway, along with benzoyl-CoA, serve as effector molecules that bind to HbdR inducing the expression of the hbd genes. Moreover, the hbd genes are subject to additional regulation influenced by the presence of non-aromatic carbon sources (carbon catabolite repression), and their expression is induced in oxygen-deprived conditions by the AcpR transcriptional activator. The prevalence of the hbd cluster among members of the Aromatoleum/Thauera bacterial group, coupled with its association with mobile genetic elements, suggests acquisition through horizontal gene transfer. These findings significantly enhance our understanding of the regulatory mechanisms governing the hbd gene cluster in bacteria, paving the way for further exploration into the anaerobic utilization/valorization of phenolic compounds derived from lignin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Multipartite genomes are thought to confer evolutionary advantages to bacteria by providing greater metabolic flexibility in fluctuating environments and enabling rapid adaptation to new ecological niches and stress conditions. This genome architecture is commonly found in plant symbionts, including nitrogen-fixing rhizobia, such as Rhizobium leguminosarum bv. trifolii TA1 (RtTA1), whose genome comprises a chromosome and four extrachromosomal replicons (ECRs). In this study, the transcriptomic responses of RtTA1 to partial nutrient limitation and low acidic pH were analyzed using high-throughput RNA sequencing. RtTA1 growth under these conditions resulted in the differential expression of 1035 to 1700 genes (DEGs), which were assigned to functional categories primarily related to amino acid and carbohydrate metabolism, ribosome and cell envelope biogenesis, signal transduction, and transcription. These results highlight the complexity of the bacterial response to stress. Notably, the distribution of DEGs among the replicons indicated that ECRs played a significant role in the stress response. The transcriptomic data align with the Rhizobium pangenome analysis, which revealed an over-representation of functional categories related to transport, metabolism, and regulatory functions on ECRs. These findings confirm that ECRs contribute substantially to the ability of rhizobia to adapt to challenging environmental conditions.

CRISPR-dead Cas9 interference (CRISPRi) has become a valuable tool for precise gene regulation. In this study, CRISPRi was designed to target the inhA gene of Mycobacterium smegmatis (Msm), a gene necessary for mycolic acid synthesis. Our findings revealed that sgRNA2 induced with 100 ng/ml aTc achieved over 90% downregulation of inhA gene expression and inhibited bacterial viability by approximately 1,000-fold. Furthermore, CRISPRi enhanced the susceptibility of M. smegmatis to isoniazid and rifampicin, which are both 50% and 90% lower than those of the wild-type strain or other strains, respectively. This study highlights the ability of CRISPRi to silence the inhA gene, which impacts bacterial viability and drug susceptibility. The findings provide valuable insights into the utility of CRISPRi as an alternative tool for gene regulation. CRISPRi might be further assessed for its synergistic effect with current anti-tuberculosis drugs and its possible implications for combating mycobacterial infections, especially drug-resistant tuberculosis., (© 2024. The Author(s).)

Brucella spp. is an intracellular bacterium that uses its transcriptional regulator DeoR1 to promote intracellular transport and survival, but the molecular mechanism remains unknown. To analyze the role of DeoR1 in the virulence of B. abortus and the genes regulated by DeoR1, we created a A19ΔdeoR1 mutant of B. abortus A19 (A19). Virulence assay was performed using a murine macrophage cell line (RAW264.7) and mice. We observed that A19ΔdeoR1 mutant is attenuated in RAW264.7 cells and mice. We performed RNA-seq whole transcriptome analysis of A19ΔdeoR1 and A19 from infected RAW264.7 cells. A total of 135 differentially expressed genes were identified, including 100 up-regulated and 35 down-regulated genes. These differentially expressed genes were involved in amino acid synthesis and metabolism, energy production and conversion, stress proteins, chaperonin, hypothetical proteins and protein of unknown function, cell wall/membrane/envelope, intracellular transporting and secretion, and transcriptional regulator. Interestingly, genes involved in the intracellular trafficking and secretion were significantly down-regulated in A19ΔdeoR1. Furthermore, selected RNA-seq results were experimentally confirmed by qRT-PCR. Overall, these results deciphered differential phenomena associated with virulence in A19ΔdeoR1 and A19 from infected RAW264.7 cells, which provided important information for understanding the detailed role of DeoR1 in Brucella pathogenesis. SIGNIFICANCE: Transcriptional regulators are predominant bacterial signal transduction factors. The pathogenicity of Brucella is due to its ability to regulate the expression of virulence related genes. Transcriptional regulators are designed to regulate gene expression and enact an appropriate adaptive physiological response. Here, a total of 135 differentially expressed genes were identified in transcriptional regulator deoR1 mutant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Three RpoD-family sigma factors, RpoD, RpoS, and RpoH, play critical roles in transcriptional regulation in Salmonella enterica serovar Typhimurium under heat shock conditions. However, the genome-wide regulatory mechanisms of these sigma factors in response to heat stress have remained elusive. In this study, we comprehensively identified 2,319, 2,226, and 213 genome-wide binding sites for RpoD, RpoS, and RpoH, respectively, under sublethal heat shock conditions (42°C). Machine learning-based transcriptome analysis was employed to infer the relative activity of iModulons, providing valuable insights into the transcriptional impact of heat shock. Integrative data analysis enabled the reconstruction of the transcriptional regulatory network of sigma factors, revealing how they modulate gene expression to adapt to heat stress, including responses to anaerobic and oxidative stresses. Notably, we observed a significant expansion of the RpoS sigmulon from 97 to 301 genes in response to heat shock, underscoring the crucial role of RpoS in regulating various metabolic processes. Moreover, we uncovered a competition mechanism between RpoD and RpoS within RpoS sigmulons, where RpoS significantly increases its binding within promoter regions shared with RpoD under heat shock conditions. These findings illuminate how three RpoD-family sigma factors coordinate multiple cellular processes to orchestrate the overall response of S. Typhimurium to heat stress., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Park et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Currently, almost all known regulators involved in bacterial phosphorus metabolism are proteins. In this study, we identified a conserved new small regulatory RNA (sRNA), named PhoS, encoded in the 3' untranslated region (UTR) of the phoPR genes in Bacillus velezensis and B. subtilis. Expression of phoS is strongly induced upon phosphorus scarcity and stimulated by the transcription factor PhoP. Conversely, PhoS positively regulates PhoP translation by binding to the ribosome binding site (RBS) of phoP mRNA. PhoS can promote Bacillus biofilm formation through, at least in part, enhancing the expression of the matrix-related genes, such as the eps genes and the tapA-sipW-tasA operon. The positive regulation of phoP expression by PhoS contributes to the promoting effect of PhoS on biofilm formation. sRNAs regulating biofilm formation have rarely been reported in gram-positive Bacillus species. Here we highlight the significance of sRNAs involved in two important biological processes: phosphate metabolism and biofilm formation., (© 2024. The Author(s).)

Pseudomonas alloputida KT2440 is a ubiquitous, soil-dwelling bacterium that metabolizes recalcitrant and volatile carbon sources. The latter is utilized by two redundant, Ca- and lanthanide (Ln)-dependent, pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ ADH), PedE and PedH, whose expression is regulated by Ln availability. P. alloputida KT2440 is the best-studied non-methylotroph in the context of Ln-utilization. Combined with microfluidic cultivation and single-cell elemental analysis, we studied the impact of light and heavy Ln on transcriptome-wide gene expression when growing P. alloputida KT2440 with 2-phenylethanol as the carbon and energy source. Light Ln (La, Ce, and Nd) and a mixture of light and heavy Ln (La, Ce, Nd, Dy, Ho, Er, and Yb) had a positive effect on growth, whereas supplementation with heavy Ln (Dy, Ho, Er, and Yb) exerted fitness costs. These were likely a consequence of mismetallation and non-utilizable Ln interfering with Ln sensing and signaling. The measured amounts of cell-associated Ln varied between elements. Gene expression analysis suggested that the Ln sensing and signaling machinery, the two-component system PedS2R2 and PedH, responds differently to (non-)utilizable Ln. We expanded our understanding of the lanthanide (Ln) switch in P. alloputida KT2440, demonstrating that it adjusts the levels of pedE and pedH transcripts based on the availability of Ln. We propose that the usability of Ln influences the bacterium's response to different Ln elements.IMPORTANCEThe Ln switch, the inverse regulation of Ca- and Ln-dependent PQQ ADH in response to Ln availability in organisms featuring both, is central to our understanding of Ln utilization. Although the preference of bacteria for light Ln is well known, the effect of different Ln, light and heavy, on growth and gene expression has rarely been studied. We provide evidence for a fine-tuning mechanism of Ca- and Ln-dependent PQQ ADH in P. alloputida KT2440 on the transcriptome level. The response to (non-)utilizable Ln differs depending on the element. Ln commonly co-occur in nature. Our findings underline that Ln-utilizing microbes must be able to discriminate between Ln to use them effectively. Considering the prevalence of Ln-dependent proteins in many microbial taxa, more work addressing Ln sensing and signaling is needed. Ln availability likely necessitates different adaptations regarding Ln utilization., Competing Interests: The authors declare no conflict of interest.

Nucleotide secondary messengers regulate various processes in bacteria allowing them to rapidly respond to changes in environmental conditions. c-di-AMP is an essential second messenger required for the growth of the human pathogen Staphylococcus aureus , regulating potassium, osmolyte uptake, and beta-lactam resistance. Cellular concentrations of c-di-AMP are regulated by the activities of two enzymes, DacA and GdpP, which synthesize and hydrolyze c-di-AMP, respectively. Besides these, only a limited number of other factors are known to regulate c-di-AMP levels. Using a c-di-AMP biosensor consisting of the Bacillus subtilis c-di-AMP-binding kimA riboswitch and yfp, we were able to efficiently detect differences in cellular c-di-AMP levels in S. aureus . To identify novel factors that regulate c-di-AMP levels, we introduced the biosensor into a library of S. aureus transposon mutants. In this manner, we obtained mutants with increased c-di-AMP levels that contained insertions in gdpP coding for the c-di-AMP hydrolase and ybbR ( cdaR ) coding for a c-di-AMP cyclase regulator, thus validating our screen. We also identified two high c-di-AMP mutants with insertions upstream of the nrdIEF operon coding for the ribonucleotide reductase enzyme. Further analysis revealed that the insertion down-regulated nrdIEF expression, indicating that the enzyme is a negative regulator of c-di-AMP production. This negative regulation was dependent on rsh , encoding for the synthase of the endogenous GdpP inhibitor (p)ppGpp. The methods established in this work can be readily adapted for use in other bacteria to uncover genetic or environmental factors regulating c-di-AMP levels.IMPORTANCEc-di-AMP is an important secondary messenger, produced by many bacterial species including the opportunistic pathogen Staphylococcus aureus . In this bacterium, c-di-AMP controls cell wall homeostasis, cell size, and osmotic balance. In addition, it has been shown that strains with high c-di-AMP levels exhibit increased resistance to beta-lactam antibiotics. Here, we developed a biosensor-based method for the rapid detection of c-di-AMP levels in S. aureus . We utilized the biosensor in a genetic screen for the identification of novel factors that impact cellular c-di-AMP. In this manner, we identified the ribonucleotide reductase as a novel factor altering cellular c-di-AMP levels and showed that reducing its expression leads to increased cellular c-di-AMP levels. As methicillin-resistant S. aureus strains are considered as a global health threat, it is important to study processes that dictate cellular c-di-AMP levels, which are associated with antibiotic resistance., Competing Interests: The authors declare no conflict of interest.

The LitR/CarH family comprises adenosyl B 12 -based photosensory transcriptional regulators that control light-inducible carotenoid production in nonphototrophic bacteria. In this study, we established a blue-green light-inducible hyperexpression system using LitR and its partner ECF-type sigma factor LitS in streptomycin-producing Streptomyces griseus NBRC 13350. The constructed multiple-copy number plasmid, pLit19, carried five genetic elements: pIJ101rep, the thiostrepton resistance gene, litR, litS, and σ LitS -recognized light-inducible crtE promoter. Streptomyces griseus transformants harboring pLit19 exhibited a light-dependent hyper-production of intracellular reporter enzymes including catechol-2,3-dioxygenase and β-glucuronidase, extracellular secreted enzymes including laccase and transglutaminase, and secondary metabolites including melanin, flaviolin, and indigoidine. Cephamycin-producing Streptomyces sp. NBRC 13304, carrying an entire actinorhodin gene cluster, exhibited light-dependent actinorhodin production after the introduction of the pLit19 shuttle-type plasmid with the pathway-specific activator actII-ORF4. Insertion of sti fragment derived from Streptomyces phaeochromogenes pJV1 plasmid into pLit19 increased its light sensitivity, allowing gene expression under weak light irradiation. The two constructed Escherichia coli-Streptomyces shuttle-type pLit19 plasmids were found to have abilities similar to those of pLit19. We successfully established an optogenetically controlled hyperproduction system for S. griseus NBRC 13350 and Streptomyces sp. NBRC 13304., (© 2024. The Author(s).)

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pseudomonas plecoglossicida is a causative agent of visceral granulomas in large yellow croaker ( Larimichthys crocea ). Quorum sensing (QS) is widely involved in imparting virulence to pathogenic bacteria; however, it has not been studied in P. plecoglossicida . In this study, we annotated a LuxR family transcriptional regulator in P. plecoglossicida NB2011 and designated as PplR. We aligned the protein sequence by BlastP and Clustal X2, monitored the N-acyl-homoserine lactone (AHL) signal production through cross-feeding bioassay and HC-MS/MS; investigated exogenous AHL signal binding by recombinant expression and thin layer chromatography; constructed a deletion mutant of the target gene by method of double homologous recombination; sequenced the transcript RNA and analyzed the data; additionally, characterized phenotypes of wild type and mutant strain. The LuxR homolog PplR was found to share high similarity with PpoR-the LuxR solo of Pseudomonas putida -without a cognate LuxI. The wild-type strain did not produce any AHL signals and the recombinant LuxR protein was found to bind C6-L-homoserine lactone (C6-HSL), C8-HSL, 3-oxo-C10-HSL, and 3-oxo-C12-HSL. RNA-seq analysis indicated 84 differentially expressed genes-5 upregulated and 79 downregulated-mainly enriched in gene ontology terms, such as flagella-dependent motility, integral component of membrane, DNA binding and transcription, and metal ion binding, suggesting that PplR is a master transcription regulator. The mutant strain showed attenuated biofilm-forming ability and stress resistance, and the data support a role for PplR in the regulation of these traits in P. plecoglossicida NB2011 independent of the presence of AHL signals. This is the first study to provide QS-related information on P. plecoglossicida ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Jia, Chi, Chen and Mao.)

Cell motility increases the fitness of bacterial cells. Previous research focused on the transcriptional regulator CdsR, which represses cellular autolysis and promotes spore formation in Bacillus thuringiensis. However, the targets of CdsR are mostly unknown. Here, we reported a new function of CdsR in regulating cell motility. Mutation of cdsR results in increase of cell mobility, and a number of related genes were upregulated compared to wild type HD73. Thus, we investigated the transcription of the fla/che gene cluster, which involves in cell mobility and comprises eight operons/genes, including motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, and yjbJ-flgG. Additionally, the motAB2 operon was discovered, which consists of homologs genes motA2 and motB2 that are like motA1 and motB1. Through promoter-lacZ fusion assays and EMSA experiments, it was discovered that CdsR directly regulates the motAB1, cheY-yrhK, lamB-cheR, yaaR-fliG2, cheV-mogR, hag1, hag2, yjbJ-flgG, and motAB2 operons by binding to their promoter regions. Importantly, it was confirmed that CdsR is a metalloregulator and the binding to promoter can be inhibited by Cu (II) ions. This research enhances our understanding of the regulation of cell mobility in B. thuringiensis., (© 2024. The Author(s).)

Bacterial two-component systems (TCSs) are signaling modules that control physiology, adaptation, and host interactions. A typical TCS consists of a histidine kinase (HK) that activates a response regulator via phosphorylation in response to environmental signals. Here, we systematically test the effect of inactivating the conserved phosphatase activity of HKs to activate TCS signaling pathways. Transcriptome analyses of 14 HK mutants in Streptococcus agalactiae, the leading cause of neonatal meningitis, validate the conserved HK phosphatase mechanism and its role in the inhibition of TCS activity in vivo. Constitutive TCS activation, independent of environmental signals, enables high-resolution mapping of the regulons for several TCSs (e.g., SaeRS, BceRS, VncRS, DltRS, HK11030, HK02290) and reveals the functional diversity of TCS signaling pathways, ranging from highly specialized to interconnected global regulatory networks. Targeted analysis shows that the SaeRS-regulated PbsP adhesin acts as a signaling molecule to activate CovRS signaling, thereby linking the major regulators of host-pathogen interactions. Furthermore, constitutive BceRS activation reveals drug-independent activity, suggesting a role in cell envelope homeostasis beyond antimicrobial resistance. This study highlights the versatility of constitutive TCS activation, via phosphatase-deficient HKs, to uncover regulatory networks and biological processes., (© 2024. The Author(s).)

Rickettsia rickettsii is an obligate intracellular, tick-borne bacterium that causes Rocky Mountain spotted fever. The demanding nature of cultivating these bacteria within host cells and the labor involved in obtaining clonal isolates have severely limited progress regarding the development of compatible genetic tools to study this pathogen. Conditional expression of genes that might be toxic or have an otherwise undesirable effect is the next logical goal to expand upon the constitutive expression plasmids generated thus far. We describe the construction of an inducible promoter system based on the tet-On system, leveraging design elements from the anhydrotetracycline-inducible promoter system used for Borrelia burgdorferi and one of the few characterized rickettsial promoters for the outer membrane gene, rompB ( sca5 ). The functionality of this promoter is demonstrated via fluorescence of induced mScarlet production and was then used to construct a generalized inducible expression vector for R. rickettsii . The development of a functional inducible promoter was then applied to the construction of a CRISPR interference plasmid as a means to reduce or essentially silence the transcription of targeted genes. We demonstrate the viability of a simplified, single vector CRISPRi system to disrupt gene expression in R. rickettsii targeting the type IV secreted effector rarP2 and autotransporter peptidase rapL as examples., Importance: This work expands upon the genetic toolbox available for R. rickettsii . We describe both an inducible promoter and CRISPRi system compatible with Rickettsia , which may provide key instruments for the development of further tools. The development of an inducible promoter system allows for the overexpression of genes, which might be toxic when expressed constitutively. The CRISPRi system enables the ability to knock down genes with specificity, and critically, genes that may be essential and could not otherwise be knocked out. These developments may provide the foundation for unlocking genetic tools for other pathogens of the order Rickettsiales, such as the Anaplasma , Orientia , and Ehrlichia for which there are currently no inducible promoters or CRISPRi platforms., Competing Interests: The authors declare no conflict of interest.

Bacteroides species are successful colonizers of the human colon and can utilize a wide variety of complex polysaccharides and oligosaccharides that are indigestible by the host. To do this, they use enzymes encoded in polysaccharide utilization loci (PULs). While recent work has uncovered the PULs required for the use of some polysaccharides, how Bacteroides utilize smaller oligosaccharides is less well studied. Raffinose family oligosaccharides (RFOs) are abundant in plants, especially legumes, and consist of variable units of galactose linked by α-1,6 bonds to a sucrose (glucose α-1-β-2 fructose) moiety. Previous work showed that an α-galactosidase, BT1871, is required for RFO utilization in Bacteroides thetaiotaomicron . Here, we identify two different types of mutations that increase BT1871 mRNA levels and improve B. thetaiotaomicron growth on RFOs. First, a novel spontaneous duplication of BT1872 and BT1871 places these genes under the control of a ribosomal promoter, driving high BT1871 transcription. Second, nonsense mutations in a gene encoding the PUL24 anti-sigma factor likewise increase BT1871 transcription. We then show that hydrolases from PUL22 work together with BT1871 to break down the sucrose moiety of RFOs and determine that the master regulator of carbohydrate utilization (BT4338) plays a role in RFO utilization in B. thetaiotaomicron . Examining the genomes of other Bacteroides species, we found homologs of BT1871 in a subset and showed that representative strains of species with a BT1871 homolog grew better on melibiose than species that lack a BT1871 homolog. Altogether, our findings shed light on how an important gut commensal utilizes an abundant dietary oligosaccharide., Importance: The gut microbiome is important in health and disease. The diverse and densely populated environment of the gut makes competition for resources fierce. Hence, it is important to study the strategies employed by microbes for resource usage. Raffinose family oligosaccharides are abundant in plants and are a major source of nutrition for the microbiota in the colon since they remain undigested by the host. Here, we study how the model commensal organism, Bacteroides thetaiotaomicron utilizes raffinose family oligosaccharides. This work highlights how an important member of the microbiota uses an abundant dietary resource., Competing Interests: The authors declare no conflict of interest.

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis , we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 is responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation and in which the connection between Wag31, PlrA, and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate the synthesis of the different layers of the mycobacterial cell wall in changing conditions., Importance: This study is performed in Mycobacterium smegmatis , which is used as a model to understand the basic physiology of pathogenic mycobacteria such as Mycobacterium tuberculosis . In this work, we examine the function and regulation of three proteins involved in regulating cell wall elongation in mycobacterial cells, which occurs at the cell tips or poles. We find that Wag31, a regulator of polar elongation, works partly through the regulation of MmpL3, a transporter of cell wall constituents and an important drug target. Our work suggests that, beyond its transport function, MmpL3 has another function in controlling cell wall synthesis broadly in response to stress., Competing Interests: The authors declare no conflict of interest.

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46 , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation.IMPORTANCEIntracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection., Competing Interests: The authors declare no conflict of interest.

The cell cycle is a fundamental process involved in bacterial reproduction and cellular differentiation. For Sinorhizobium meliloti , cell cycle outcomes depend on its growth environment. This bacterium shows a tight coupling of DNA replication initiation with cell division during free-living growth. In contrast, it undergoes a novel program of endoreduplication and terminal differentiation during symbiosis within its host. While several DivK regulators at the top of its CtrA pathway have been shown to play an important role in this differentiation process, there is a lack of resolution regarding the downstream molecular activities required and whether they could be unique to the symbiosis cell cycle. The DivK kinase CbrA is a negative regulator of CtrA activity and is required for successful symbiosis. In this work, spontaneous symbiosis suppressors of Δ cbrA were identified as alleles of divL and cckA . In addition to rescuing symbiotic development, they restore wild-type cell cycle progression to free-living Δ cbrA cells. Biochemical characterization of the S. meliloti hybrid histidine kinase CckA in vitro demonstrates that it has both kinase and phosphatase activities. Specifically, CckA on its own has autophosphorylation activity, and phosphatase activity is induced by the second messenger c-di-GMP. Importantly, the CckA A373S suppressor protein of Δ cbrA has a significant loss in kinase activity, and this is predicted to cause decreased CtrA activity in vivo . These findings deepen our understanding of the CbrA regulatory pathway and open new avenues for further molecular characterization of a network pivotal to the free-living cell cycle and symbiotic differentiation of S. meliloti .IMPORTANCE Sinorhizobium meliloti is a soil bacterium able to form a nitrogen-fixing symbiosis with certain legumes, including the agriculturally important Medicago sativa . It provides ammonia to plants growing in nitrogen-poor soils and is therefore of agricultural and environmental significance as this symbiosis negates the need for industrial fertilizers. Understanding mechanisms governing symbiotic development is essential to either engineer a more effective symbiosis or extend its potential to non-leguminous crops. Here, we identify mutations within cell cycle regulators and find that they control cell cycle outcomes during both symbiosis and free-living growth. As regulators within the CtrA two-component signal transduction pathway, this study deepens our understanding of a regulatory network shaping host colonization, cell cycle differentiation, and symbiosis in an important model organism., Competing Interests: The authors declare no conflict of interest.

We identified and characterized genomic regions of Streptococcus agalactiae that are involved in the Leloir and the tagatose-6-phosphate pathways for D-galactose catabolism. The accumulation of mutations in genes coding the Leloir pathway and the absence of these genes in a significant proportion of the strains suggest that this pathway may no longer be necessary for S. agalactiae and is heading toward extinction. In contrast, a genomic region containing genes coding for intermediates of the tagatose-6-phosphate pathway, a Gat family PTS transporter, and a DeoR/GlpR family regulator is present in the vast majority of strains. By deleting genes that code for intermediates of each of these two pathways in three selected strains, we demonstrated that the tagatose-6-phosphate pathway is their sole route for galactose catabolism. Furthermore, we showed that the Gat family PTS transporter acts as the primary importer of galactose in S. agalactiae . Finally, we proved that the DeoR/GlpR family regulator is a repressor of the tagatose-6-phosphate pathway and that galactose triggers the induction of this biochemical mechanism.IMPORTANCE S. agalactiae , a significant pathogen for both humans and animals, encounters galactose and galactosylated components within its various ecological niches. We highlighted the capability of this bacterium to metabolize D-galactose and showed the role of the tagatose-6-phosphate pathway and of a PTS importer in this biochemical process. Since S. agalactiae relies on carbohydrate fermentation for energy production, its ability to uptake and metabolize D-galactose could enhance its persistence and its competitiveness within the microbiome., Competing Interests: The authors declare no conflict of interest.