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As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid ( Euprymna scolopes ) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri . The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V . fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions., Competing Interests: The authors declare no conflict of interest.

Ultraviolet radiation (UV) is a major abiotic stress resulting in relative short duration of Bacillus thuringiensis (Bt) biopesticides in the field, which is expected to be solved by formation of Bt biofilm with higher UV resistance. Therefore, one of the important prerequisite works is to clarify the functions of biofilm-associated genes on biofilm formation and UV resistance of Bt. In this study, comparative genomics and bioinformatic analysis indicated that BTXL6_19475 gene involved in biofilm formation of Bt XL6 was likely to encode a galactose-1-phosphate uridylyltransferase (GalT, E.C. 2.7.7.12). Heterologous expression of the BTXL6_19475 gene in Escherichia coli and detection of its GalT enzyme activity in vitro proved that the gene did encode GalT. Comparing the wild type Bt strain XL6 with galT gene knockout mutant Bt XL6ΔgalT and its complementary strain Bt XL6ΔgalT::19,475, GalT promoted the biofilm formation and enhanced the UV-B resistance of Bt XL6 likely by increasing its D-ribose production and reducing its alanine aryldamidase activity. GalT did not affect the growth and the cell motility of Bt XL6. A regulation map had been proposed to elucidate how GalT promoted biofilm formation and enhanced UV-B resistance of Bt XL6 by the cross-talk between Leloir pathway, Embden-Meyerhof glycolysis pathway and pentose phosphate pathway. Our finding provides a theoretical basis for the efficient use of biofilm genes to improve the UV resistance of Bt biofilms and thus extend field duration of Bt formulations based on biofilm engineering., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN-binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process., (© 2024 Eschbach et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

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).)

Transcription elongation is one of the most important processes in the cell. During RNA polymerase elongation, the folding of nascent transcripts plays crucial roles in the genetic decision. Bacterial riboswitches are prime examples of RNA regulators that control gene expression by altering their structure upon metabolite sensing. It was previously revealed that the thiamin pyrophosphate-sensing tbpA riboswitch in Escherichia coli cotranscriptionally adopts three main structures leading to metabolite sensing. Here, using single-molecule FRET, we characterize the transition in which the first nascent structure, a 5' stem-loop, is unfolded during transcription elongation to form the ligand-binding competent structure. Our results suggest that the structural transition occurs in a relatively abrupt manner, i.e., within a 1-2 nucleotide window. Furthermore, a highly dynamic structural exchange is observed, indicating that riboswitch transcripts perform rapid sampling of nascent co-occurring structures. We also observe that the presence of the RNAP stabilizes the 5' stem-loop along the elongation process, consistent with RNAP interacting with the 5' stem-loop. Our study emphasizes the role of early folding stem-loop structures in the cotranscriptional formation of complex RNA molecules involved in genetic regulation., 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 Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger in bacteria that regulates multiple biological functions, including biofilm formation, virulence, and intercellular communication. However, c-di-GMP signaling is virtually unknown in economically important filamentous cyanobacteria, Arthrospira. In this study, we predicted 31 genes encoding GGDEF-domain proteins from A. platensis NIES39 as potential diguanylate cyclases (DGCs). Phylogenetic distribution analysis showed five genes (RS09460, RS04865, RS26155, M01840, and E02220) with highly conserved distribution across 25 Arthrospira strains. Adc1 encoded by RS09460 was further characterized as a typical DGC. By establishing the genetic transformation system of Arthrospira, we demonstrated that the overexpression of Adc1 promoted the production of extracellular polymeric substances (EPS), which in turn caused the aggregation of filaments. We also confirmed that RS04865 and RS26155 may encode active DGCs, while enzymatic activity assays showed that proteins encoded by M01840 and E02220 have phosphodiesterase (PDE) activity. Meta-analysis revealed that the expression profiles of RS09460 and RS04865 were unaffected under 31 conditions, suggesting that they may function as conserved genes in maintaining the basal level of c-di-GMP in Arthrospira. In summary, this report will provide the basis for further studies of c-di-GMP signal in Arthrospira., 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.)

The multifunctional proteins of class 2 CRISPR systems such as Cas9, have been employed to activate cryptic biosynthetic gene clusters (BGCs) in Streptomyces, which represent a large and hidden reservoir of natural products. However, such approaches are not applicable to most Streptomyces strains with reasons to be comprehended. Inspired by the prevalence of the class 1 subtype especially the type I-E CRISPR system in Streptomyces, here we report the development of the type I-E CRISPR system into a series of transcriptional regulation tools. We further demonstrate the effectiveness of such activators in nine phylogenetically distant Streptomyces strains. Using these tools, we successfully activate 13 out of 21 BGCs and lead to the identification and characterization of one polyketide, one Ripp and three alkaloid products. Our work is expected to have a profound impact and to facilitate the discovery of numerous structurally diverse compounds from Streptomyces., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The functions of non-coding small RNAs (sRNAs) within the human microbiome remain largely unexplored. In this Cell Host & Microbe issue, El Mouali et al. identify Segatella RNA colonization factor (SrcF), a sRNA from a prevalent gut bacterium Segatella copri. SrcF promotes colonization of S. copri by regulating bacterial degradation of complex dietary carbohydrates., Competing Interests: Declaration of interests M.S.D. works as a consultant and an advisory board member at Theralution GmbH, Germany., (Copyright © 2024 Elsevier Inc. 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.

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δ fhs and Δ proABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δ fhs and Δ proABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 Δ proABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 Δ proABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δ fhs and Δ proABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δ fhs had an impaired stringent response, and when cultured in sera, BHN418 Δ fhs and Δ proABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae . We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential., Competing Interests: The authors declare no conflict of interest.

The bacterium Segatella copri is a prevalent member of the human gut microbiota associated with health and disease states. However, the intrinsic factors that determine its ability to colonize the gut effectively remain largely unknown. By extensive transcriptome mapping of S. copri and examining human-derived samples, we discover a small RNA, which we name Segatella RNA colonization factor (SrcF), and show that SrcF is essential for S. copri gut colonization in gnotobiotic mice. SrcF regulates genes involved in nutrient acquisition, and complex carbohydrates, particularly fructans, control its expression. Furthermore, SrcF expression is strongly influenced by human microbiome composition and by the breakdown of fructans by cohabitating commensals, suggesting that the breakdown of complex carbohydrates mediates interspecies signaling among commensals beyond its established function in generating energy. Together, this study highlights the contribution of a small RNA as a critical regulator in gut colonization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gene expression and proper downstream cellular functions upon facing environmental shifts depend on the combined and cooperative regulation of genetic networks. Here, we identified cAMP receptor protein (CRP) as a master regulator of (p)ppGpp (guanosine tetra- and penta-phosphate) homeostasis. Via CRP-mediated direct transcriptional regulation of the (p)ppGpp synthetase/hydrolase RelA and SpoT, cAMP-CRP stimulates pervasive accumulation of (p)ppGpp under glucose-limiting conditions. Notably, CRP exerts a nonclassical property as a translational regulator through YfiQ-dependent acetylation of ribosome protein S1 at K247, which further enhances the translation of RelA, SpoT, and CRP itself. From a synthetic biology perspective, this self-activating feedback loop for (p)ppGpp synthesis highlights the function of C RP- m ediated d ual e nhancement (CMDE) in controlling bacterial gene expression, which enables stable activation of genetic circuits. CMDE applied in synthetic circuits leads to a stable increase in p -coumaric acid, cinnamic acid, and pinosylvin production. Our findings showed that CRP-mediated dual circuits for (p)ppGpp regulation enable robust activation that could address bioproduction and other biotechnological needs.IMPORTANCETranscriptional-translational coordination is fundamental for rapid and efficient gene expression in most bacteria. Here, we uncovered the roles of cAMP-CRP in this process. We found that CRP distinctly increases RelA and SpoT transcription and translation, and that acetylation of S1 at K247 accelerates the self-activation of the leading CRP under glucose-limiting conditions. We further found that elevated (p)ppGpp significantly impedes the formation of the cAMP-CRP complex, an active form responsible for transcriptional activation. A model was created in which cAMP-CRP and (p)ppGpp cooperate to dynamically modulate the efficiency of transcriptional-translational coordination responses to stress. More broadly, productive activation in synthetic circuits was achieved through the application of C RP- m ediated d ual e nhancement (CMDE), promising to inspire new approaches for the development of cell-based biotechnologies., Competing Interests: The authors declare no conflict of interest.

Opportunistic fish pathogen Vibrio scophthalmi frequently infects olive flounder and turbot, which are primary marine species cultured for seafood production in Far East Asia. These infections cause substantial yield reductions and significant economic losses. Although quorum sensing (QS) genes were previously reported in V. scophthalmi, the impacts of QS on genome-wide gene expression and consequent behaviors and physiological traits have remained largely unexplored. In this study, we conducted genomic and transcriptomic analyses to uncover the global regulatory network governed by LuxR Vs , a QS master regulator in V. scophthalmi. By comparing the wild-type strain and a luxR Vs deletion mutant strain, we found that LuxR Vs positively regulates biosynthetic genes for poly-hydroxyalkanoate (PHA) while negatively controlling genes for biofilm formation. Quantification of intracellular PHAs and biofilm biomass on borosilicate tubes confirmed these results. Gene set enrichment analyses further demonstrated that LuxR Vs also governs genes related to osmoprotection and defense against reactive oxygen species. Overall, these findings indicate that LuxR Vs acts as a global transcriptional regulator, controlling a wide range of physiological processes in V. scophthalmi. Targeting LuxR Vs could therefore be a promising strategy for improving seafood production by disrupting diverse physiological and pathogenic traits in this fish pathogen., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

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 toxin-antitoxin (TA) system regulates many physiological processes in free-living bacteria. One such TA system in Escherichia coli comprises an RNA toxin SdsR and an antitoxin RyeA. An overabundance of SdsR is toxic to the cells. RyeA normalizes SdsR abundance and helps the cells to adapt to altered conditions. The current study showed that a novel small RNA (sRNA) regulator GcvB directly interacts with RyeA to maintain its abundance in the cells under normal or low pH conditions. The deletion of the gcvB allele in the E. coli chromosome resulted in a ∼3-fold decrease in intrabacterial RyeA accumulation. An ectopic expression of GcvB in ΔgcvB strain reinstated RyeA abundance to its normal level. Induction of GcvB in the cells upon exposure to low pH resulted in a simultaneous increase in intracellular RyeA. While GcvB increases RyeA abundance in the cells, SdsR accumulation is divergently regulated by GcvB. The absence of the gcvB gene in E. coli leads to upregulation of SdsR and vice versa. The GcvB-mediated decrease of SdsR accumulation stems from the increased RyeA-driven normalization of SdsR. This study delineates a novel mechanism for the regulation of the expression of an RNA toxin SdsR by another sRNA regulator GcvB through a feed-forward control., 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 Inc. All rights reserved.)

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.)

Type IV CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA sequencing (RNA-seq) analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range downregulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Diazotrophic cyanobacteria can fix nitrogen gas (N 2 ) but are usually scarce in nitrogen-limited coastal waters, which poses an apparent ecological paradox. One hypothesis is that high salinities (> 10 g/L NaCl) may inhibit cyanobacterial N 2 fixation. However, here we show that N 2 fixation in a unicellular coastal cyanobacterium exclusively depends on sodium ions and is inhibited at low NaCl concentrations (< 4 g/L). In the absence of Na + , cells of Cyanothece sp. ATCC 51142 (recently reclassified as Crocosphaera subtropica) upregulate the expression of nifHDK genes and synthesise a higher amount of nitrogenase, but do not fix N 2 and do not grow. We find that the loss of N 2 -fixing ability in the absence of Na + is due to insufficient ATP supply. Additional experiments suggest that N 2 fixation in this organism is driven by sodium energetics and mixed-acid fermentation, rather than proton energetics and aerobic respiration, even though cells were cultured aerobically. Further work is needed to clarify the underlying mechanisms and whether our findings are relevant to other coastal cyanobacteria., (© 2024. The Author(s).)

During growth, cells need to synthesize and expand their envelope, a process that requires careful regulation. Here, we show that the GTPase ObgE of E. coli contributes to the regulation of lipopolysaccharide (LPS) synthesis, an essential component of the Gram-negative outer membrane. Using a dominant-negative mutant (named 'ObgE*'), we show a direct interaction between ObgE and LpxA, which catalyzes the first step in LPS synthesis. This interaction is enhanced by the mutation in ObgE* which, when bound to GTP, leads to inhibition of LpxA, decreased LPS synthesis, and cell death. Although wild-type ObgE does not exert the same strong effects as ObgE* on LpxA or LPS synthesis, our data indicate that ObgE participates in the regulation of cell envelope synthesis in E. coli. Because ObgE also influences other cellular functions (i.e., ribosome assembly, DNA replication, etc.), it seems increasingly plausible that this GTPase coordinates several processes to finetune cell growth., (© 2024. The Author(s).)

Cell growth and gene expression are heterogeneous processes at the single-cell level, leading to the emergence of multiple physiological states within bacterial populations. Aging is a known deterministic driver of growth asymmetry; however, its role in gene expression heterogeneity remains elusive. Here, we show that aging mother cells undergo a progressive decline in old pole activity, generating asymmetry in protein partitioning, gene expression, and cell morphology. We demonstrate that mother cells, when compared to their daughters, exhibit lower product inheritance and gene expression rates independently of promoter dynamics. The declining activity of maternal old poles generates gene expression gradients that manifest as mother-daughter asymmetry upon division, showing that asymmetry is progressively built over time within the maternal intracellular environment. Moreover, old pole aging correlates with a gradual increase in cell length, leading to morphological asymmetry. These findings provide further evidence for aging as a mechanism to enhance phenotypic heterogeneity in bacterial populations, with possible consequences for stress response and survival.

Antimicrobial resistance (AMR) is a significant global threat, and the presence of resistance-determinant genes is one of the major driving forces behind it. The bacterial rod complex is an essential set of proteins that is crucial for cell survival due to its role in cell wall biogenesis and shape maintenance. Therefore, these proteins offer excellent potential as drug targets; however, compensatory mutations in nontarget genes render this complex nonessential. The MreB protein of this complex is an actin homologue that rotates along the longitudinal axis of the cell to provide rod shape to the bacteria. In this study, using chemical-chemical interaction profiling and FtsZ suppression assay, we identified the MreB targeting activity of IITR07865, a previously discovered small molecule in our lab. Escherichia coli suppressors against IITR07865 revealed mutations in two cell division-associated genes, min C and pal , that have not been previously implicated in rod complex essentiality. IITR07865 resistant mutants were found to inactivate and render the rod complex nonessential, making the rod complex inhibitors ineffective. Further, through transcriptome analysis, we reveal the primary cause of resistance in suppressor strains to be the overexpression of an l, d-transpeptidase A enzyme, which is involved in peptidoglycan and Braun's lipoprotein cross-linking. Our results demonstrate a novel mechanism of resistance development in rod-shaped Gram-negative bacterial pathogen E. coli involved in UTIs where mecillinam, a clinically used antibiotic that targets rod complex, is a drug of choice.

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).)

We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the hag locus, a σD-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter, following a deletion of the gene encoding a post-translational regulator of σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics., Competing Interests: CH, VR, BS, JO, and ZA are paid employees of ZBiotics Company and hold stock/stock options in ZBiotics Company. ZA is also a board member of ZBiotics Company. As such, employees of ZBiotics played a role in the study design, data collection and analysis, decision to publish, and preparation of the manuscript. Investors in ZBiotics Company provided financial support to the company and thus supported the salaries of CH, VR, BS, JO, and ZA but did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Hassan-Casarez 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.)

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cA n ) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA 4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA 4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity., Competing Interests: Declaration of interests G. Tamulaitis is an inventor on patents and patent applications related to Csm and CARF protein applications., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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).)

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as P T5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

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.

The trade-off between rapid growth and other important physiological traits (e.g., survival and adaptability) poses a fundamental challenge for microbes to achieve fitness maximization. Studies on Bacillus subtilis biology often use strains derived after a process of lab 'domestication' from an ancestral strain known as Marburg strain. The domestication process led to loss of a large plasmid (pBS32) encoding a phosphatase (RapP) that dephosphorylates the Spo0F protein and thus regulates biofilm formation and sporulation. Here, we show that plasmid pBS32, and more specifically rapP, enhance growth rates by preventing premature expression of the Spo0F-Spo0A-mediated adaptive response during exponential phase. This results in reallocation of proteome resources towards biosynthetic, growth-promoting pathways without compromising long-term fitness during stationary phase. Thus, RapP helps B. subtilis to constrain physiological trade-offs and economize cellular resources for fitness improvement., (© 2024. The Author(s).)

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.

Streptococcus agalactiae (or group B Streptococcus , GBS) is a leading cause of neonatal sepsis and meningitis globally. To sense and respond to variations in its environment, GBS possesses multiple two-component regulatory systems (TCSs), such as LytSR. Here, we aimed to investigate the role of LytSR in GBS pathogenicity. We generated an isogenic lytS knockout mutant in a clinical GBS isolate and used a combination of phenotypic in vitro assays and in vivo murine models to investigate the contribution of lytS to the colonization and invasive properties of GBS. Deletion of the lytS gene in the GBS chromosome resulted in significantly higher survival rates in mice during sepsis, accompanied by reduced bacterial loads in blood, lung, spleen, kidney, and brain tissues compared to infection with the wild-type strain. In a mouse model of GBS vaginal colonization, we also observed that the lytS knockout mutant was cleared more readily from the vaginal tract compared to its wild-type counterpart. Interestingly, lower levels of proinflammatory cytokines were found in the serum of mice infected with the lytS mutant. Our results demonstrate that the LytSR TCS plays a key role in GBS tissue invasion and pathogenesis, and persistence of mucosal colonization.IMPORTANCE Streptococcus agalactiae ( group B Streptococcus, or GBS) is a common commensal of the female urogenital tract and one of WHO's priority pathogens. The bacterium has evolved mechanisms to adapt and survive in its host, many of which are regulated via two-component signal transduction systems (TCSs); however, the exact contributions of TCSs toward GBS pathogenicity remain largely obscure. We have constructed a TCS lytS- deficient mutant in a CC-17 hypervirulent GBS clinical isolate. Using murine models, we showed that LytSR regulatory system is essential for vaginal colonization via promoting biofilm production. We also observed that lytS deficiency led to significantly attenuated virulence properties and lower levels of proinflammatory cytokines in blood. Our findings are of significant importance in that they unveil a previously unreported role for LytSR in GBS and pave the way toward a better understanding of its ability to transition from an innocuous commensal to a deadly pathogen., Competing Interests: Marie O'Brien is an employee and co-founder of ReNewVax Ltd.

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.

Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. In this study, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium Escherichia coli K-12 as well as strains of the antibiotic-resistant pathogen, Acinetobacter baumannii . We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design. Embedded in these vectors, we also developed a synthetic, IPTG-inducible promoter, P abstBR , that induces to a high level but is less leaky than the commonly used trc promoter. We show that P abstBR is titratable at both the population and single-cell levels, regardless of species, highlighting the utility of our expression systems for cross-species functional studies. Finally, as a proof of principle, we use our integrating vector to develop a reporter for the E. coli envelope stress σ factor, RpoE, and deploy the reporter in E. coli and A. baumannii , finding that A. baumannii does not recognize RpoE-dependent promoters unless RpoE is heterologously expressed. We envision that these vector and promoter tools will be valuable for the community of researchers who study the fundamental biology of E. coli and A. baumannii .IMPORTANCE Acinetobacter baumannii is a multidrug-resistant, hospital-acquired pathogen with the ability to cause severe infections. Understanding the unique biology of this non-model bacterium may lead to the discovery of new weaknesses that can be targeted to treat antibiotic-resistant infections. In this study, we provide expression tools that can be used to study the gene function in A. baumannii , including in drug-resistant clinical isolates. These tools are also compatible with the model bacterium, Escherichia coli , enabling cross-species comparisons of gene function. We anticipate that the use of these tools by the scientific community will accelerate our understanding of Acinetobacter biology., Competing Interests: The authors declare no conflict of interest.

Campylobacter jejuni is a major cause of food- and water-borne bacterial infections in humans. A key factor helping bacteria to survive adverse environmental conditions is biofilm formation ability. Nonetheless, the molecular basis underlying biofilm formation by C. jejuni remains poorly understood. Around thirty genes involved in the regulation and dynamics of C. jejuni biofilm formation have been described so far. We applied random transposon mutagenesis to identify new biofilm-associated genes in C. jejuni strain 81-176. Of 1350 mutants, twenty-four had a decreased ability to produce biofilm compared to the wild-type strain. Some mutants contained insertions in genes previously reported to affect the biofilm formation process. The majority of identified genes encoded hypothetical proteins. In the library of EZ-Tn5 insertion mutants, we found the cydB gene associated with respiration that was not previously linked with biofilm formation in Campylobacter. To study the involvement of the cydB gene in biofilm formation, we constructed a non-marked deletion cydB mutant together with a complemented mutant. We found that the cydB deletion-mutant formed a weaker biofilm of loosely organized structure and lower volume than the parent strain. In the present study, we demonstrated the role of the cydB gene in biofilm formation by C. jejuni., (© 2024. The Author(s).)

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).)

Salmonella Typhimurium (STM), a zoonotic pathogen, can adjust its metabolic pathway according to the variations in the partial pressure of atmospheric oxygen and nitrate via fumarate nitrate reductase regulator (Fnr) and NarL, the response regulator for nitrate reductase. Both Fnr and NarL have been individually reported to be the contributors of virulent phenotypes of STM. Hypoxia along with nitrate-rich environment are prevalent in macrophages and the Salmonella-induced inflammatory lumen of the host's large intestine activates both fnr and narL genes. In this study, the double (fnr and narL) knockout STM showed a synergistic reduction in the swimming (62%), swarming (84%) and biofilm density (86%) phenotypes anaerobically in association with its significant aerobic attenuation. The intracellular replication of the double mutant was reduced by 2.3 logs in chicken monocyte-derived macrophages. Furthermore, the competitive index of the double mutant in liver and spleen was found to be 0.3 and 0.44 respectively at 120 h post-infection (PI) in mice. Surprisingly, no double mutant could be recovered from the infected mouse liver 3 days PI. Histopathological findings showed moderate infiltration of mononuclear cells in the large intestine of mice infected with double mutant, but severe infiltration was seen with the wild-type strain., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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.)

Escherichia coli, a common microbial host for industrial bioproduction, experiences a highly dynamic environment in industrial-scale bioreactors due to significant glucose and oxygen gradients. In this study, we mimic the combined gradients of glucose and oxygen in high-throughput bioreactors to study the transcriptional response of E. coli to industrial-scale conditions. Under oscillating oxygen conditions, E. coli formed less biomass and accumulated the anaerobic by-product acetate. With respect to oxygen-responsive genes, we found that genes of the TCA cycle and of different electron transport chain complexes were differentially expressed. A global analysis of the expression data revealed that oxygen oscillations had caused a transition towards a catabolite-repressed state and upregulation of several stress-related regulatory programs. Interestingly, the transcriptional changes persisted after oxygen limitation stopped. In contrast, the changes we observed due to glucose starvation, such as induction of the stringent response, were primarily transient. Most importantly, we found that effects of combined oxygen and glucose oscillations were distinct from the ones of oxygen and substrate oscillations alone, suggesting an important interplay between the different metabolic regimes in industrial-scale bioreactors., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Bacteria employ two-component systems (TCSs) to rapidly sense and respond to their surroundings often and during plant infection. Poplar canker caused by Lonsdalea populi is an emerging woody bacterial disease that leads to high mortality and poplar plantation losses in China. Nonetheless, the information about the underlying mechanism of pathogenesis remains scarce. Therefore, in this study, we reported the role of a TCS pair CpxA/CpxR in regulating virulence and stress responses in L. populi. The CpxA/R system is essential during infection, flagellum formation, and oxidative stress response. Specifically, the Cpx system affected flagellum formation by controlling the expression of flagellum-related genes. CpxR, which was activated by phosphorylation in the presence of CpxA, participated in the transcriptional regulation of a chaperone sctU and the type III secretion system (T3SS)-related genes, thereby influencing T3SS functions during L. populi infection. Phosphorylated CpxR directly manipulated the transcription of a membrane protein-coding gene yccA and the deletion of yccA resulted in reduced virulence and increased sensitivity to H 2 O 2 . Furthermore, we mutated the conserved phosphorylation site of CpxR and found that CpxR D51A could no longer bind to the yccA promoter but could still bind to the sctU promoter. Together, our findings elucidate the roles of the Cpx system in regulating virulence and reactive oxygen species resistance and provide further evidence that the TCS is crucial during infection and stress response., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

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.

Lignocellulolytic clostridia employ multiple pairs of alternative σ/anti-σ (SigI/RsgI) factors to regulate cellulosomal components for substrate-specific degradation of cellulosic biomass. The current model has proposed that RsgIs use a sensor domain to bind specific extracellular lignocellulosic components and activate cognate SigIs to initiate expression of corresponding cellulosomal enzyme genes, while expression of scaffoldins can be initiated by several different SigIs. Pseudobacteroides cellulosolvens contains the most complex known cellulosome system and the highest number of SigI-RsgI regulons yet discovered. However, the function of many RsgI sensor domains and their relationship with the various enzyme types are not fully understood. Here, we report that RsgI4 from P. cellulosolvens employs a C-terminal module that bears distant similarity to the fibronectin type III (Fn3) domain and serves as the sensor domain. Substrate-binding analysis revealed that the Fn3-like domain of RsgI4 represents a novel carbohydrate-binding module (CBM) that binds to a wide range of polysaccharide types. Structure determination further revealed that the Fn3-like domain belongs to the type B group of CBMs with a predicted concave face for substrate binding. Promoter sequence analysis of cellulosomal genes revealed that SigI4 is responsible for cellulosomal regulation of major scaffoldins rather than enzymes, consistent with the broad substrate specificity of the RsgI4 sensor domain. Notably, scaffoldins are invariably required as cellulosome components regardless of the substrate type. These findings suggest that the intricate cellulosome system of P. cellulosolvens comprises a more elaborate regulation mechanism than other bacteria and thus expands the paradigm of cellulosome regulation., (© 2024 The Protein Society.)

The Type VI secretory system (T6SS) is a key regulatory network in the bacterial system, which plays an important role in host-pathogen interactions and maintains cell homeostasis by regulating the release of effector proteins in specific competition. T6SS causes cell lysis or competitive inhibition by delivering effector molecules, such as toxic proteins and nucleic acids, directly from donor bacterial cells to eukaryotic or prokaryotic targets. Additionally, it orchestrates synthesis of immune effectors that counteract toxins thus preventing self-intoxication or antagonistic actions by competing microbes. Even so, the mechanism of toxin-antitoxin regulation in bacteria remains unclear. In response, this review discusses the bacterial T6SS's structure and function and the mechanism behind toxin-antitoxin secretion and the T6SS's expression in order to guide the further exploration of the pathogenic mechanism of the T6SS and the development of novel preparations for reducing and replacing toxins and antitoxins., 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.)

The transition from planktonic to biofilm growth in bacteria is often accompanied by greater resistance to antibiotics and other stressors, as well as distinct alterations in physical traits, genetic activity, and metabolic restructuring. In many species, the heme nitric oxide/oxygen binding proteins (H-NOX) play an important role in this process, although the signaling mechanisms and pathways in which they participate are quite diverse and largely unknown. In Paracoccus denitrificans , deletion of the hnox gene results in a severe biofilm-deficient phenotype. Quantitative proteomics was used to assemble a comprehensive data set of P. denitrificans proteins showing altered abundance of those involved in several important metabolic pathways. Further, decreased levels of pyruvate and elevated levels of C 16 homoserine lactone were detected for the Δhnox strain, associating the biofilm deficiency with altered central carbon metabolism and quorum sensing, respectively. These results expand our knowledge of the important role of H-NOX signaling in biofilm formation.

Vibrio cholerae is an inherent inhabitant of aquatic ecosystems. The Indian state of West Bengal, especially the Gangetic delta region is the highest cholera affected region and is considered as the hub of Asiatic cholera. V. cholerae were isolated from publicly accessible wastewater of Midnapore, West Bengal, India. Serotyping determined all isolates to be of non-O1/non-O139 serogroups. Moderate biofilm-forming abilities were noticed in most of the isolates (74.7 %) while, high biofilm formation was recorded for only 6.3 % isolates and 19 % of isolates exhibited low/non-biofilm-forming abilities. PCR-based screening of crucial diguanylate cyclases (DGCs) involved in cyclic-di-GMP-mediated biofilm signaling was performed. cdgH and cdgM were the most abundant DGCs among 93.7 % and 91.5 % of isolates, respectively. Other important DGCs, i.e., cdgK, cdgA, cdgL, and vpvC were present in 84 %, 75.5 %, 72 % and 68 % of isolates, respectively. Besides, the non-O1/non-O139 isolates were screened for the occurrence of virulence factor encoding genes. Moreover, among these non-O1/non-O139 isolates, two strains (3.17 %) harbored both ctxA and ctxB genes, which encode the cholera toxin associated with epidemic cholera. ompU was the most prevalent virulence factor, present in 24.8 % of isolates. Other virulence factors like, zot and st were found in 4.7 % and 9.5 % of isolates. Genes encoding tcp and ace were found to be PCR-negative for the isolates. Additionally, crucial virulence factor regulators, toxT, toxR and hapR were found to be PCR-positive in all the isolates. Antibiotic resistance patterns displayed further vulnerabilities with decreased sensitivity towards commonly used antibiotics with multiple antibiotic resistance index ranging between 0.37 and 0.62. The presence of cholera toxin-encoding multi-drug resistant (MDR) V. cholerae strains in environmental settings is alarming. High occurrence of DGCs are considered to encourage further investigations to use them as alternative therapeutic targets against MDR cholera pathogen due to their unique presence in bacterial systems., 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.)