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The central carbon/lactate utilization pathway in the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, is encoded by the highly conserved operon DVU3025-3033. Our earlier in vitro genome-wide study had suggested a network of four two-component system regulators that target this large operon; however, how these four regulators control this operon was not known. Here, we probe the regulation of the lactate utilization operon with mutant strains and DNA-protein binding assays. We show that the LurR response regulator is required for optimal growth and complete lactate utilization, and that it activates the DVU3025-3033 lactate oxidation operon as well as DVU2451, a lactate permease gene, in the presence of lactate. We show by electrophoretic mobility shift assays that LurR binds to three sites in the upstream region of DVU3025, the first gene of the operon. NrfR, a response regulator that is activated under nitrite stress, and LurR share similar binding site motifs and bind the same sites upstream of DVU3025. The DVU3025 promoter also has a binding site motif (Pho box) that is bound by PhoB, a two-component response regulator activated under phosphate limitation. The lactate utilization operon, the regulator LurR, and LurR binding sites are conserved across the order Desulfovibrionales whereas possible modulation of the lactate utilization genes by additional regulators such as NrfR and PhoB appears to be limited to D. vulgaris.

BackgroundThe human pathogen Corynebacterium diphtheriae is the causative agent of diphtheria. In the 1990s a large diphtheria outbreak in Eastern Europe was caused by the strain C. diphtheriae NCTC 13129. Although the genome was sequenced more than a decade ago, not much is known about its transcriptome. Our aim was to use transcriptome sequencing (RNA-Seq) to close this knowledge gap and gain insights into the transcriptional landscape of a C. diphtheriae tox+ strain.ResultsWe applied two different RNA-Seq techniques, one to retrieve 5'-ends of primary transcripts and the other to characterize the whole transcriptional landscape in order to gain insights into various features of the C. diphtheriae NCTC 13129 transcriptome. By examining the data we identified 1656 transcription start sites (TSS), of which 1202 were assigned to genes and 454 to putative novel transcripts. By using the TSS data promoter regions recognized by the housekeeping sigma factor σA and its motifs were analyzed in detail, revealing a well conserved -10 but an only weakly conserved -35 motif, respectively. Furthermore, with the TSS data 5'-UTR lengths were explored. The observed 5'-UTRs range from zero length (leaderless transcripts), which make up 20% of all genes, up to over 450 nt long leaders, which may harbor regulatory functions. The C. diphtheriae transcriptome consists of 471 operons which are further divided into 167 sub-operon structures. In a differential expression analysis approach, we discovered that genetic disruption of the iron-sensing transcription regulator DtxR, which controls expression of diphtheria toxin (DT), causes a strong influence on general gene expression. Nearly 15% of the genome is differentially transcribed, indicating that DtxR might have other regulatory functions in addition to regulation of iron metabolism and DT. Furthermore, our findings shed light on the transcriptional landscape of the DT encoding gene tox and present evidence for two tox antisense RNAs, which point to a new way of transcriptional regulation of toxin production.ConclusionsThis study presents extensive insights into the transcriptome of C. diphtheriae and provides a basis for future studies regarding gene characterization, transcriptional regulatory networks, and regulation of the tox gene in particular.

The Pss-I region of Rhizobium leguminosarum bv. trifolii TA1 comprises more than 20 genes coding for glycosyltransferases, modifying enzymes, and polymerization/export proteins, altogether determining the biosynthesis of symbiotically relevant exopolysaccharides. In this study, the role of homologous PssG and PssI glycosyltransferases in exopolysaccharide subunit synthesis were analyzed. It was shown that the glycosyltransferase-encoding genes of the Pss-I region were part of a single large transcriptional unit with potential downstream promoters activated in specific conditions. The ΔpssG and ΔpssI mutants produced significantly lower amounts of the exopolysaccharide, while the double deletion mutant ΔpssIΔpssG produced no exopolysaccharide. Complementation of double mutation with individual genes restored exopolysaccharide synthesis, but only to the level similar to that observed for the single ΔpssI or ΔpssG mutants, indicating that PssG and PssI serve complementary functions in the process. PssG and PssI interacted with each other in vivo and in vitro. Moreover, PssI displayed an expanded in vivo interaction network comprising other GTs involved in subunit assembly and polymerization/export proteins. PssG and PssI proteins were shown to interact with the inner membrane through amphipathic helices at their C-termini, and PssG also required other proteins involved in exopolysaccharide synthesis to localize in the membrane protein fraction. [ABSTRACT FROM AUTHOR]

Cytochrome P450 monooxygenases (CYPs/P450s) and their redox partners, ferredoxins, are ubiquitous in organisms. P450s have been studied in biology for over six decades owing to their distinct catalytic activities, including their role in drug metabolism. Ferredoxins are ancient proteins involved in oxidation-reduction reactions, such as transferring electrons to P450s. The evolution and diversification of P450s in various organisms have received little attention and no information is available for archaea. This study is aimed at addressing this research gap. Genome-wide analysis revealed 1204 P450s belonging to 34 P450 families and 112 P450 subfamilies, where some families and subfamilies are expanded in archaea. We also identified 353 ferredoxins belonging to the four types 2Fe-2S, 3Fe-4S, 7Fe-4S and 2[4Fe-4S] in 40 archaeal species. We found that bacteria and archaea shared the CYP109, CYP147 and CYP197 families, as well as several ferredoxin subtypes, and that these genes are co-present on archaeal plasmids and chromosomes, implying the plasmid-mediated lateral transfer of these genes from bacteria to archaea. The absence of ferredoxins and ferredoxin reductases in the P450 operons suggests that the lateral transfer of these genes is independent. We present different scenarios for the evolution and diversification of P450s and ferredoxins in archaea. Based on the phylogenetic analysis and high affinity to diverged P450s, we propose that archaeal P450s could have diverged from CYP109, CYP147 and CYP197. Based on this study's results, we propose that all archaeal P450s are bacterial in origin and that the original archaea had no P450s. [ABSTRACT FROM AUTHOR]

The proximity of arsenic in environment has transformed into a genuine natural issue among various regions of the world. An assortment of microorganisms is recognized today remediating arsenic by their metabolic pathways. Arsenicals like arsenate and arsenite are misleadingly similar to vital growth supplements. Structurally arsenate resembles to phosphate, competitively enters the cell through phosphate transportation channels and aquaglyceroporins (arsenite) respectively. The aox gene array were identified and distinguished for conveying arsenite oxidation using oxidases. Native microscopic organisms catalyze arsenic transformation encouraged by anaerobic condition through arr and ars operon systems. The genes for arsenic resistance were identified by nucleotide sequencing and the existence of multiple operon systems (arsRBCDA) were confirmed. The orf arrangements as an operon is utilized as a part of arsenic resistance mechanism by microorganisms residing in tainted mining regions. Many microorganisms are also identified harboring multi-operon arsenic resisting/transforming operons, including operon switching property according to the environment. Likewise, many other genes are also discovered to play important role in metabolizing arsenic, discussed here. This study concentrates on thrusts in biochemical and molecular developments in the field of arsenic metabolizing microbial systems, covering concept of redox operon systems and their mechanism of action. The molecular investigations of these microorganisms and their proteins may prompt development of fruitful bioremediation techniques. [ABSTRACT FROM AUTHOR]

In bacteria, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we could not identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene argX, (coding tRNAarg) from Brevibacterium albidum, and we transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE, galT, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. Since the quantitative real-time PCR (qRT-PCR) method detects the steady state following mRNA synthesis and degradation, we observed that tRNAarg is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg can directly reflect the mRNA synthesis. Using this approach, we were able to effectively assay the RDTs and Rho-independent termination (RIT) in the gal operon by quantifying the relative amount of tRNAarg using qRT-PCR analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. The resultant RIT% at the end of the gal operon is 33%. Our findings demonstrate that combining tRNAarg with qRT-PCR can directly measure RIT, RDT, or any other signal that attenuates transcription efficiencies in vivo, making it a useful tool for gene expression research.

Chloroviruses (family Phycodnaviridae) are dsDNA viruses found throughout the world's inland waters. The open reading frames in the genomes of 41 sequenced chloroviruses (330 ± 40 kbp each) representing three virus types were analyzed for evidence of evolutionarily conserved local genomic "contexts", the organization of biological information into units of a scale larger than a gene. Despite a general loss of synteny between virus types, we informatically detected a highly conserved genomic context defined by groups of three or more genes that we have termed "gene gangs". Unlike previously described local genomic contexts, the definition of gene gangs requires only that member genes be consistently co-localized and are not constrained by strand, regulatory sites, or intervening sequences (and therefore represent a new type of conserved structural genomic element). An analysis of functional annotations and transcriptomic data suggests that some of the gene gangs may organize genes involved in specific biochemical processes, but that this organization does not involve their coordinated expression.

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

Cellular thiols such as cysteine spontaneously and readily react with the respiratory intermediate fumarate, resulting in the formation of stable S-(2-succino)-adducts. Fumarate-mediated succination of thiols increases in certain tumors and in response to glucotoxicity associated with diabetes. Therefore, S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC) are considered oncometabolites and biomarkers for human disease. No disposal routes for S-(2-succino)-compounds have been reported prior to this study. Here, we show that Bacillus subtilis metabolizes 2SC to cysteine using a pathway encoded by the yxe operon. The first step is N-acetylation of 2SC followed by an oxygenation that we propose results in the release of oxaloacetate and N-acetylcysteine, which is deacetylated to give cysteine. Knockouts of the genes predicted to mediate each step in the pathway lose the ability to grow on 2SC as the sulfur source and accumulate the expected upstream metabolite(s). We further show that N-acetylation of 2SC relieves toxicity. This is the first demonstration of a metabolic disposal route for any S-(2-succino)-compound, paving the way toward the identification of corresponding pathways in other species.

Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.

Fusion constructs as protein overexpression vectors proved to be critical in the heterologous expression of terpene synthases in cyanobacteria. The concept was recently applied to the heterologous overexpression of the β-phellandrene synthase (β- PHLS) from plants, fused to the highly expressed endogenous cpcB gene encoding the β-subunit of phycocyanin. Overexpressed CpcB*PHLS fusion proteins enhanced the heterologous yield of C10H16 β-phellandrene hydrocarbons production in Synechocystis. This work extended the concept of fusion constructs as protein overexpression vectors by showing that highly expressed heterologous genes could also serve as leader sequences for protein overexpression in cyanobacteria. Examined are the kanamycin nptI and chloramphenicol cmR resistance cassettes, both of which are overexpressed in Synechocystis. Evidence showed a dual purpose of the nptI gene, as a leader sequence fused to a heterologous geranyl-diphosphate synthase ( GPPS), promoting its expression, while at the same time serving as a selectable marker for the screening of transformants. The work further showed that enhanced GPPS expression increased the yield of β-phellandrene in Synechocystis transformants harboring the β- PHLS gene. Moreover, the research evaluated the expression efficacy of a DNA fragment comprising 87 nucleotides from the 5' end of the cmR gene in fusion with the GPPS gene. This short fusion construct substantially increased the intracellular geranyl-diphosphate synthase level, suggesting that "short-stretch" cmR leader sequences can be used to drive a higher expression level of heterologous biosynthetic genes, while avoiding undesirable internal recombinations, as these sequences are shorter than the threshold of 200 bp, commonly assumed to be the threshold of high efficiency recombinations.

Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium, Pseudomonas stutzeri RCH2, by three two-component regulatory proteins CopR1, CopR2 and CzcR. We conducted genome-wide evaluations to identify gene targets of two paralogous regulators, CopR1 and CopR2, annotated for copper signaling, and compared the results with the gene targets for CzcR, implicated in zinc signaling. We discovered that the CopRs and CzcR have largely common targets, and crossregulate a core set of P. stutzeri copper and zinc responsive genes. We established that this crossregulation is enabled by a conserved binding motif in the upstream regulatory regions of the target genes. The crossregulation is physiologically relevant as these regulators synergistically and antagonistically target multicopper oxidases, metal efflux and sequestration systems. CopR1 and CopR2 upregulate two cop operons encoding copper tolerance genes, while all three regulators downregulate a putative copper chaperone, Psest_1595. CzcR also upregulated the oprD gene and the CzcIABC Zn2+ efflux system, while CopR1 and CopR2 downregulated these genes. Our study suggests that crossregulation of copper and zinc homeostasis can be advantageous, and in P. stutzeri this is enabled by shared binding motifs for multiple response regulators.

The gram‐negative bacterium Erwinia amylovora causes fire blight disease of apple and pear trees. The exopolysaccharide amylovoran and lipopolysaccharides are essential E. amylovora virulence factors. Production of amylovoran and lipopolysaccharide is specified in part by genes that are members of long operons. Here, we show that full virulence of E. amylovora in apple fruitlets and tree shoots depends on the predicted transcription antiterminator RfaH. RfaH reduces pausing in the production of long transcripts having an operon polarity suppressor regulatory element within their promoter region. In E. amylovora, only the amylovoran operon and a lipopolysaccharide operon have such regulatory elements within their promoter regions and in the correct orientation. These operons showed dramatically increased polarity in the ΔrfaH mutant compared to the wild type as determined by RNA sequencing. Amylovoran and lipopolysaccharide production in vitro was reduced in rfaH mutants compared to the wild type, which probably contributes to the rfaH mutant virulence phenotype. Furthermore, type VI secretion cluster 1, which contributes to E. amylovora virulence, showed reduced expression in ΔrfaH compared to the wild type, although without an increase in polarity. The data suggest that E. amylovora RfaH directly, specifically, and exclusively suppresses operon polarity in the amylovoran operon and a lipopolysaccharide operon. [ABSTRACT FROM AUTHOR]

Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. This discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.

2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. 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.)

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).)

Levan biopolymer and levan-type fructooligosaccharides (L-FOSs) are β-2,6-linked fructans that have been used as non-digestible dietary fiber and prebiotic oligosaccharides in food and cosmeceutical applications. In this study, we explore the operon responsible for levan and L-FOSs production in Priestia koreensis HL12. Presented is the first genomic perspective on sucrose utilization and the levan biosynthesis pathway in this bacterium. Regarding sequence annotation, the putative levansucrase operon responsible for β-2,6-linked fructan was identified in the genome of strain HL12, and comprises sacB levansucrase gene belonging to GH68, located adjacent to levB endo-levanase gene, which belongs to GH32. Importantly, sugars related with the levan biosynthesis pathway are proposed to be transported via 3 types of transportation systems, including multiple ABC Sugar and glucose/H + transporters, as well as glucose- and fructose-specific PTS systems. Based on product profile analysis, the HL12 strain exhibited high efficiency in levan production from high sucrose concentration (300 g/l), achieving the highest yield of 127 g/l (equivalent to 55% conversion based on sucrose consumption), together with short-chain L-FOSs (DP3-5) and long-chain L-FOSs with respective size larger than DP6 after 48 h incubation. These findings highlight the potential of P. koreensis HL12 as a whole-cell biocatalyst for producing levan and L-FOSs, and underscore its novelty in converting sugars into high-value-added products for diverse commercial and industrial applications.

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.

Limiting the availability of transition metals at infection sites serves as a critical defense mechanism employed by the innate immune system to combat microbial infections. Pseudomonas aeruginosa exhibits a remarkable ability to thrive in zinc-deficient environments, facilitated by intricate cellular responses governed by numerous genes regulated by the zinc-responsive transcription factor Zur. Many of these genes have unknown functions, including those within the predicted PA2911-PA2914 and PA4063-PA4066 operons. A structural bioinformatics investigation revealed that PA2911-PA2914 comprises a TonB-dependent outer membrane receptor and inner membrane ABC-permeases responsible for importing metal-chelating molecules, whereas PA4063-PA4066 contains genes encoding a MacB transporter, likely involved in the export of large molecules. Molecular genetics and biochemical experiments, feeding assays, and intracellular metal content measurements support the hypothesis that PA2911-PA2914 and PA4063-PA4066 are engaged in the import and export of the pyochelin-cobalt complex, respectively. Notably, cobalt can reduce zinc demand and promote the growth of P. aeruginosa strains unable to import zinc, highlighting pyochelin-mediated cobalt import as a novel bacterial strategy to counteract zinc deficiency. These results unveil an unexpected role for pyochelin in zinc homeostasis and challenge the traditional view of this metallophore exclusively as an iron transporter., Importance: The mechanisms underlying the remarkable ability of Pseudomonas aeruginosa to resist the zinc sequestration mechanisms implemented by the vertebrate innate immune system to control bacterial infections are still far from being fully understood. This study reveals that the Zur-regulated gene clusters PA2911-2914 and PA4063-PA4066 encode systems for the import and export of cobalt-bound pyochelin, respectively. This proves to be a useful strategy to counteract conditions of severe zinc deficiency since cobalt can replace zinc in many proteins. The discovery that pyochelin may contribute to cellular responses to zinc deficiency leads to a reevaluation of the paradigm that pyochelin is a siderophore involved exclusively in iron acquisition and suggests that this molecule has a broader role in modulating the homeostasis of multiple metals., Competing Interests: The authors declare no conflict of interest.

The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase) was replaced with that of P hrtB , creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a fully automated laboratory platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant. Reversely engineered strains showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter in bacteria. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production., (© 2024. The Author(s).)

Mycobacterium tuberculosis has evolved a highly specialized system to snatch essential nutrients from its host, among which host-derived cholesterol has been established as one main carbon source for M. tuberculosis to survive within granulomas. The uptake, catabolism, and utilization of cholesterol are important for M. tuberculosis to sustain within the host largely via remodeling of the bacterial cell walls. However, the regulatory mechanism of cholesterol uptake and its impact on bacterium fate within infected hosts remain elusive. Here, we found that M. tuberculosis LacI-type transcription regulator Rv3575c negatively regulates its mce4 family gene transcription. Overexpression of Rv3575c impaired the utilization of cholesterol as the sole carbon source by Mycobacterium smegmatis , activating the host's innate immune response and triggering cell pyroptosis. The M. smegmatis homologue of Rv3575c MSMEG6044 knockout showed enhanced hydrophobicity and permeability of the cell wall and resistance to ethambutol, suppressed the host innate immune response to M. smegmatis , and promoted the survival of M. smegmatis in macrophages and infected mouse lungs, leading to reduced transcriptional levels of TNFα and IL-6. In summary, these data indicate a role of Rv3575c in the pathogenesis of mycobacteria and reveal the key function of Rv3575c in cholesterol transport in mycobacteria.

Aims: Klebsiella michiganensis is a medically important bacterium that has been subject to relatively little attention in the literature. Interrogation of sequence data from K. michiganensis strains in our collection has revealed the presence of multiple large plasmids encoding type II toxin-antitoxin (TA) systems. Such TA systems are responsible for mediating a range of phenotypes, including plasmid stability ('addiction') and antibiotic persistence. In this work, we characterize the hipBA TA locus found within the Klebsiella oxytoca species complex (KoSC)., Methods and Results: The HipBA TA system is encoded on a plasmid carried by K. michiganensis PS_Koxy4, isolated from an infection outbreak. Employing viability and plasmid stability assays, we demonstrate that PS_Koxy4 HipA is a potent antibacterial toxin and that HipBA is a functional TA module contributing substantially to plasmid maintenance. Further, we provide in silico data comparing HipBA modules across the entire KoSC., Conclusions: We provide the first evidence of the role of a plasmid-encoded HipBA system in stability of mobile genetic elements and analyse the presence of HipBA across the KoSC. These results expand our knowledge of both a common enterobacterial TA system and a highly medically relevant group of bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

ICE KKS102 Tn 4677 carries a bph operon for the mineralization of polychlorinated biphenyls (PCBs)/biphenyl and belongs to the Tn 4371 ICE (integrative and conjugative element) family. In this study, we investigated the role of the traR gene in ICE transfer. The traR gene encodes a LysR-type transcriptional regulator, which is conserved in sequence, positioning, and directional orientation among Tn 4371 family ICEs. The traR belongs to the bph operon, and its overexpression on solid medium resulted in modest upregulation of traG (threefold), marked upregulation of xis (80-fold), enhanced ICE excision and, most notably, ICE transfer frequency. We propose the evolutional roles of traR , which upon insertion to its current position, might have connected the cargo gene activation and ICE transfer. This property of ICE, i.e., undergoing transfer under environmental conditions that lead to cargo gene activation, would instantly confer fitness advantages to bacteria newly acquiring this ICE, thereby resulting in efficient dissemination of the Tn 4371 family ICEs.IMPORTANCEOnly ICE KKS102 Tn 4677 is proven to transfer among the widely disseminating Tn 4371 family integrative and conjugative elements (ICEs) from β and γ-proteobacteria. We showed that the traR gene in ICE KKS102 Tn 4677 , which is conserved in the ICE family with fixed location and direction, is co-transcribed with the cargo gene and activates ICE transfer. We propose that capturing of traR by an ancestral ICE to the current position established the Tn 4371 family of ICEs. Our findings provide insights into the evolutionary processes that led to the widespread distribution of the Tn 4371 family of ICEs across bacterial species., Competing Interests: The authors declare no conflict of interest.

In E. coli K-12, the absence of unphosphorylated PtsN (unphospho-PtsN) has been proposed to cause an L-leucine-sensitive growth phenotype (Leu S ) by hyperactivated K + uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L-valine (Val)-sensitive acetohydroxyacid synthase I (AHAS I) that renders residual AHAS activity susceptible to inhibition by Leu and K + . This leads to AHAS insufficiency and a requirement for L-isoleucine (Ile). Herein, we provide an alternate mechanism for the Leu S of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the Leu S indicate that impaired expression of the ilvBN operon jointly caused by the absence of unphospho-PtsN and the presence of Leu coupled to Leu-mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K + uptake and an elevated α-ketobutyrate level mediate elevation of ilvBN expression and alleviate the Leu S . The requirement of unphospho-PtsN as a positive regulator of ilvBN expression may buffer Ile biosynthesis against Leu-mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realized in certain environments that impair AHAS function., (© 2024 John Wiley & Sons Ltd.)

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon. Complete or partial deletions of the operon establish interdependent relationships among GV proteins during assembly. We also examine the tolerance of the GV assembly process to protein mutations and the cellular burdens caused by GV proteins. Clusters of GV protein interactions are revealed, proposing plausible protein complexes that are important for GV assembly. We anticipate our findings will set the stage for designing GVs that efficiently assemble in heterologous hosts during biomedical applications., (© 2024. The Author(s).)

The rapid expansion of whole genome sequencing in bacterial taxonomy has revealed deep evolutionary relationships and speciation signals, but assembly methods often miss true nucleotide diversity in the ribosomal operons. Though it lacks sufficient phylogenetic signal at the species level, the 16S ribosomal RNA gene is still much used in bacterial taxonomy. In cyanobacterial taxonomy, comparisons of 16S-23S Internal Transcribed Spacer (ITS) regions are used to bridge this information gap. Although ITS rRNA region analyses are routinely being used to identify species, researchers often do not identify orthologous operons, which leads to improper comparisons. No method for delineating orthologous operon copies from paralogous ones has been established. A new method for recognizing orthologous ribosomal operons by quantifying the conserved paired nucleotides in a helical domain of the ITS, has been developed. The D1' Index quantifies differences in the ratio of pyrimidines to purines in paired nucleotide sequences of this helix. Comparing 111 operon sequences from 89 strains of Brasilonema, four orthologous operon types were identified. Plotting D1' Index values against the length of helices produced clear separation of orthologs. Most orthologous operons in this study were observed both with and without tRNA genes present. We hypothesize that genomic rearrangement, not gene duplication, is responsible for the variation among orthologs. This new method will allow cyanobacterial taxonomists to utilize ITS rRNA region data more correctly, preventing erroneous taxonomic hypotheses. Moreover, this work could assist genomicists in identifying and preserving evident sequence variability in ribosomal operons, which is an important proxy for evolution in prokaryotes., (© 2024 The Author(s). Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

Resource competition is a driver of gut microbiota composition. Bacteria can outcompete metabolically similar rivals through the limitation of shared growth-fuelling nutrients. The mechanisms underlying this remain unclear for bacteria with identical sets of metabolic genes. Here we analysed the lactose utilization operon in the murine commensal Escherichia coli 8178. Using in vitro and in vivo approaches, we showed that translation of the lactose utilization repressor gene lacI from its native non-canonical GTG start codon increases the basal expression of the lactose utilization cluster, enhancing adaptation to lactose consumption. Consequently, a strain carrying the wild type lacI GTG start codon outperformed the lacI ATG start codon mutant in the mouse intestine. This advantage was attenuated upon limiting host lactose intake through diet shift or altering the mutant frequency, emphasizing the context-dependent effect of a single nucleotide change on the bacterial fitness of a common member of the gut microbiota. Coupled with a genomic analysis highlighting the selection of non-ATG start codons in sugar utilization regulator genes across the Enterobacteriaceae family, our data exposed an unsuspected function of non-canonical start codons in metabolic competition., (© 2024. The Author(s).)

D-galactonate, a widely prevalent sugar acid, was first reported as a nutrient source for enteric bacteria in the 1970s. Since then, decades of research enabled a description of the modified Entner-Doudoroff pathway involved in its degradation and reported the structural and biochemical features of its metabolic enzymes, primarily in Escherichia coli K-12. However, only in the last few years, the D-galactonate transporter has been characterized, and the regulation of the dgo operon, encoding the structural genes for the transporter and enzymes of D-galactonate metabolism, has been detailed. Notably, in recent years, multiple evolutionary studies have identified the dgo operon as a dominant target for adaptation of E. coli in the mammalian gut. Despite considerable research on dgo operon, numerous fundamental questions remain to be addressed. The emerging relevance of the dgo operon in host-bacterial interactions further necessitates the study of D-galactonate metabolism in other enterobacterial strains., 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.)

Two new species of Dulcicalothrix, D. adhikaryi sp. nov. and D. iyengarii sp. nov., were discovered in India and are characterized and described in accordance with the rules of the International Code of Nomenclature for algae, fungi, and plants (ICN). As a result of phylogenetic analysis, Calothrix elsteri is reassigned to Brunnivagina gen. nov. During comparison with all Dulcicalothrix for which sequence data were available, we observed that the genus has six ribosomal operons in three orthologous types. Each of the three orthologs could be identified based upon indels occurring in the D1-D1' helix sequence in the ITS rRNA region between the 16S and 23S rRNA genes, and in these three types, there were operons containing ITS rRNA regions with and without tRNA genes. Examination of complete genomes in Dulcicalothrix revealed that, at least in the three strains for which complete genomes are available, there are five ribosomal operons, two with tRNA genes and three with no tRNA genes in the ITS rRNA region. Internal transcribed spacer rRNA regions have been consistently used to differentiate species, both on the basis of secondary structure and percent dissimilarity. Our findings call into question the use of ITS rRNA regions to differentiate species in the absence of efforts to obtain multiple operons of the ITS rRNA region through cloning or targeted PCR amplicons. The ITS rRNA region data for Dulcicalothrix is woefully incomplete, but we provide herein a means for dealing with incomplete data using the polyphasic approach to analyze diverse molecular character sets. Caution is urged in using ITS rRNA data, but a way forward through the complexity is also proposed., (© 2024 The Author(s). Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

Mobile genetic elements (MGEs) enable bacteria to acquire novel genes and traits. However, the functions of cargo genes within MGEs remain poorly understood. The cadmium resistance operon cadDX is present in many gram-positive bacteria. Although cadDX has been reported to be involved in metal detoxification, its regulatory mechanisms and functions in bacterial pathogenesis are poorly understood. This study revealed that cadDX contributes to cadmium resistance, oxidative stress resistance, and virulence in Streptococcus suis, an important zoonotic pathogen in pigs and humans. CadX represses cadD expression by binding to the cadDX promoter. Notably, cadX responds to H 2 O 2 stress through an additional promoter within the cadDX operon, mitigating the harmful effect of excessive cadD expression during oxidative stress. cadDX resides within an 11 K integrative and mobilizable element that can autonomously form circular structures. Moreover, cadDX is found in diverse MGEs, accounting for its widespread distribution across various bacteria, especially among pathogenic streptococci. Transferring cadDX into another zoonotic pathogen, Streptococcus agalactiae, results in similar phenotypes, including resistance to cadmium and oxidative stresses and increased virulence of S. agalactiae in mice. The new functions and regulatory mechanisms of cadDX shed light on the importance of the cadDX system in driving evolutionary adaptations and survival strategies across diverse gram-positive bacteria., (© 2024. The Author(s).)

Escherichia coli expresses surface appendages including fimbriae, flagella, and curli, at various levels in response to environmental conditions and external stimuli. Previous studies have revealed an interplay between expression of fimbriae and flagella in several E. coli strains, but how this regulation between fimbrial and flagellar expression affects adhesion to interfaces is incompletely understood. Here, we investigate how the concurrent expression of fimbriae and flagella by engineered strains of E. coli MG1655 affects their adhesion at liquid-solid and liquid-liquid interfaces. We tune fimbrial and flagellar expression on the cell surface through plasmid-based inducible expression of the fim operon and fliC-flhDC genes. We show that increased fimbrial expression increases interfacial adhesion as well as bacteria-driven actuation of micron-sized objects. Co-expression of flagella in fimbriated bacteria, however, does not greatly affect either of these properties. Together, these results suggest that interfacial adhesion as well as motion actuated by adherent bacteria can be altered by controlling the expression of surface appendages.

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata Glutamic acid Over-Producing, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program. We identify Rut sites in 5'-untranslated regions of key CSR genes/operons (cspA, cspB, cspG, and nsrR-rnr-yjfHI) that trigger premature RDTT at 37°C but not at 15°C. High concentrations of RNA chaperone CspA or nucleotide changes in the cspA mRNA leader reduce RDTT efficiency, revealing how RNA restructuring directs Rho to activate CSR genes during the cold shock and to silence them during cold acclimation. These findings establish a paradigm for how RNA thermosensors can modulate gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Halomonas elongata OUT30018 is a moderately halophilic bacterium that synthesizes and accumulates ectoine as an osmolyte by activities of the enzymes encoded by the high salinity-inducible ectABC operon. Previously, we engineered a γ-aminobutyric acid (GABA)-producing H. elongata GOP-Gad (Δ ectABC::mCherry-HopGadmut ) from an ectoine-deficient mutant of this strain due to its ability to use high-salinity biomass waste as substrate. Here, to further increase GABA accumulation, we deleted gabT, which encodes GABA aminotransferase (GABA-AT) that catalyzes the first step of the GABA catabolic pathway, from the H. elongata GOP-Gad genome. The resulting strain H. elongata ZN3 (Δ ectABC::mCherry-HopGadmut Δ gabT ) accumulated 291 µmol/g cell dry weight (CDW) of GABA in the cells, which is a 1.5-fold increase from H. elongata GOP-Gad's 190 µmol/g CDW. This result has confirmed the role of GABA-AT in the GABA catabolic pathway. However, redundancy in endogenous GABA-AT activity was detected in a growth test, where a gabT -deletion mutant of H. elongata OUT30018 was cultured in a medium containing GABA as the sole carbon and nitrogen sources. Because L-2,4-diaminobutyric acid aminotransferase (DABA-AT), encoded by an ectB gene of the ectABC operon, shares sequence similarity with GABA-AT, a complementation analysis of the gabT and the ectB genes was performed in the H. elongata ZN3 genetic background to test the involvement of DABA-AT in the redundancy of GABA-AT activity. Our results indicate that the expression of DABA-AT can restore GABA-AT activity in H. elongata ZN3 and establish DABA-AT's aminotransferase activity toward GABA in vivo ., Importance: In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing H. elongata ZN3 strain by deleting the gabT gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first in vivo evidence for GABA aminotransferase activity of an ectB -encoded DABA-AT, confirming a longstanding speculation based on the reported in vitro GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the ectB gene is upregulated., Competing Interests: The authors declare no conflict of interest.

Hydrogen sulfide (H 2 S), mainly produced from L-cysteine (Cys), renders bacteria highly resistant to oxidative stress and potentially increases antimicrobial resistance (AMR). CyuR is a Cys-dependent transcription regulator, responsible for the activation of the cyuPA operon and generation of H 2 S. Despite its potential importance, its regulatory network remains poorly understood. In this study, we investigate the roles of the CyuR regulon in a Cys-dependent AMR mechanism in E. coli strains. We show: (1) Generation of H 2 S from Cys affects the sensitivities to growth inhibitors; (2) Cys supplementation decreases stress responses; (3) CyuR negatively controls the expression of mdlAB encoding a potential transporter for antibiotics; (4) CyuR binds to a DNA sequence motif 'GAAwAAATTGTxGxxATTTsyCC' in the absence of Cys; and (5) CyuR may regulate 25 additional genes which were not reported previously. Collectively, our findings expand the understanding of the biological roles of CyuR relevant to antibiotic resistance associated with Cys., (© 2024. The Author(s).)

Species within the Campylobacter genus are recognized as emerging human pathogens. Common to all known members of the genus is the presence of an asparagine-linked glycosylation pathway encoded by the pgl operon. Campylobacter species are divided into two major groups, Group I and Group II. To date, most biochemical studies have focused on the Group I species including Campylobacter jejuni . We recently reported that the Group II Campylobacter concisus pathway deviates from that of Group I by the inclusion of a C-6″-oxidized GalNAc (GalNAcA) at the third position installed by PglJ. Herein, we investigate the diversification of the PglH enzymes that act subsequent to installation of GalNAcA. The majority of pgl operons from Group II species, including C. concisus , encode two GT-B fold glycosyltransferases (GTs), PglH1 and PglH2. As the functions of these GTs were not clear by simple comparison of their sequences to that of C. jejuni PglH, further analyses were required. We show that subsequent to the action of PglJ, PglH2 installs the next HexNAc followed by PglH1 adding a single sugar. These steps diverge from the C. jejuni pathway not only in the identity of the sugar donors (UDP-GlcNAc) but also in installing single sugars rather than acting processively. These biochemical studies were extended via bioinformatics to identify sequence signatures that provide predictive capabilities for unraveling the prokaryotic glycan landscape. Phylogenetic analysis showed early divergence between the C. jejuni PglH orthologs and C. concisus PglH1/PglH2 orthologs, leading to diversification of the final glycan.

Pseudomonas poae PMA22 produces safracins, a family of compounds with potent broad-spectrum anti-bacterial and anti-tumor activities. The safracins' biosynthetic gene cluster (BGC sac) consists of 11 ORFs organized in two divergent operons ( sacABCDEFGHK and sacIJ ) that are controlled by P a and P i promoters. Contiguous to the BGC sac, we have located a gene that encodes a putative global regulator of the LysR family annotated as MexT that was originally described as a transcriptional activator of the MexEF-OprN multidrug efflux pump in Pseudomonas . Through both in vitro and in vivo experiments, we have demonstrated the involvement of the dual regulatory system MexT-MexS on the BGC sac expression acting as an activator and a repressor, respectively. The MexEF-OprN transport system of PMA22, also controlled by MexT, was shown to play a fundamental role in the metabolism of safracin. The overexpression of mexEF-oprN in PMA22 resulted in fourfold higher production levels of safracin. These results illustrate how a pleiotropic regulatory system can be critical to optimizing the production of tailored secondary metabolites, not only through direct interaction with the BGC promoters, but also by controlling their transport.

Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3'UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD_1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3'UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus., (© 2024. The Author(s).)

Mycobacterium tuberculosis (MTB) is a pathogen that is known for its ability to persist in harsh environments and cause chronic infections. Understanding the regulatory networks of MTB is crucial for developing effective treatments. Small regulatory RNAs (sRNAs) play important roles in gene expression regulation in all kingdoms of life, and their classification based solely on genomic location can be imprecise due to the computational-based prediction of protein-coding genes in bacteria, which often neglects segments of mRNA such as 5'UTRs, 3'UTRs, and intercistronic regions of operons. To address this issue, our study simultaneously discovered genomic features such as TSSs, UTRs, and operons together with sRNAs in the M. tuberculosis H37Rv strain (ATCC 27294) across multiple stress conditions. Our analysis identified 1,376 sRNA candidates and 8,173 TSSs in MTB, providing valuable insights into its complex regulatory landscape. TSS mapping enabled us to classify these sRNAs into more specific categories, including promoter-associated sRNAs, 5'UTR-derived sRNAs, 3'UTR-derived sRNAs, true intergenic sRNAs, and antisense sRNAs. Three of these sRNA candidates were experimentally validated using 3'-RACE-PCR: predictedRNA_0240, predictedRNA_0325, and predictedRNA_0578. Future characterization and validation are necessary to fully elucidate the functions and roles of these sRNAs in MTB. Our study is the first to simultaneously unravel TSSs and sRNAs in MTB and demonstrate that the identification of other genomic features, such as TSSs, UTRs, and operons, allows for more accurate and specific classification of sRNAs., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Sugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes., Results: In this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP., Conclusions: Despite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications., (© 2024. The Author(s).)

The metabolic state of bacteria significantly contributes to their resistance to antibiotics; however, the specific metabolic mechanisms conferring antimicrobial resistance in Helicobacter pylori remain largely understudied. Employing transcriptomic and non-targeted metabolomics, we characterized the metabolic reprogramming of H. pylori when challenged with antibiotic agents. We observed a notable increase in both genetic and key proteomic components involved in fatty acid biosynthesis. Inhibition of this pathway significantly enhanced the antibiotic susceptibility of the sensitive and multidrug-resistant H. pylori strains while also disrupting their biofilm-forming capacities. Further analysis revealed that antibiotic treatment induced a stringent response, triggering the expression of the hp0560-hp0557 operon regulated by Sigma28 (σ 28 ). This activation in turn stimulated the fatty acid biosynthetic pathway, thereby enhancing the antibiotic tolerance of H. pylori . Our findings reveal a novel adaptive strategy employed by H. pylori to withstand antibiotic stress., Competing Interests: The authors declare no conflict of interest.

The hox operon in Synechocystis sp. PCC 6803, encoding bidirectional hydrogenase responsible for H 2 production, is transcriptionally upregulated under microoxic conditions. Although several regulators for hox transcription have been identified, their dynamics and higher-order DNA structure of hox region in microoxic conditions remain elusive. We focused on key regulators for the hox operon: cyAbrB2, a conserved regulator in cyanobacteria, and SigE, an alternative sigma factor. Chromatin immunoprecipitation sequencing revealed that cyAbrB2 binds to the hox promoter region under aerobic conditions, with its binding being flattened in microoxic conditions. Concurrently, SigE exhibited increased localization to the hox promoter under microoxic conditions. Genome-wide analysis revealed that cyAbrB2 binds broadly to AT-rich genome regions and represses gene expression. Moreover, we demonstrated the physical interactions of the hox promoter region with its distal genomic loci. Both the transition to microoxic conditions and the absence of cyAbrB2 influenced the chromosomal interaction. From these results, we propose that cyAbrB2 is a cyanobacterial nucleoid-associated protein (NAP), modulating chromosomal conformation, which blocks RNA polymerase from the hox promoter in aerobic conditions. We further infer that cyAbrB2, with altered localization pattern upon microoxic conditions, modifies chromosomal conformation in microoxic conditions, which allows SigE-containing RNA polymerase to access the hox promoter. The coordinated actions of this NAP and the alternative sigma factor are crucial for the proper hox expression in microoxic conditions. Our results highlight the impact of cyanobacterial chromosome conformation and NAPs on transcription, which have been insufficiently investigated., Competing Interests: RK, TO No competing interests declared, (© 2024, Kariyazono and Osanai.)

The low CO 2 -inducible NDH complex (NDH-1MS) plays a crucial role in the cyanobacterial CO 2 -concentrating mechanism. However, the components in this complex and the regulation mechanism are still not completely understood. Using a mutant only with NDH-1MS as active Ci sequestration system, we identified a functional gene sll1736 named as cupAR (CupA Regulator). The cupAR deletion mutant, ΔcupAR, grew faster than the WT under high CO 2 (HC) condition, more evidently at low pH. The activities of O 2 evolution, CO 2 uptake，NDH-1, and the building up of a transthylakoid proton were stimulated in this mutant under HC conditions. The cupAR gene is cotranscribed with the NDH-1S operon (ndhF3-ndhD3-cupA) and encoded protein, which specifically suppresses the transcription level of this operon under HC conditions. Mutation of cupAR significantly upregulated the accumulation of CupA, the key protein of NDH-1MS, under HC condition. CupAR interacted with NdhD3 and NdhF3, the membrane components of NDH-1MS, while accumulation of CupAR was reduced in the ΔndhD3 mutant. Furthermore, CupAR was colocated with CupA in both NDH-1MS complex and NDH-1S subcomplex. On the other hand, deletion of ndhR, a negative regulator of the NDH-1S operon, increased the accumulation of CupAR, whereas deletion of cupAR significantly lowered NdhR. Based on these results, we concluded that CupAR is a novel subunit of NDH-1MS, negatively regulating the activities of CupA and CO 2 uptake dependent on NDH-1MS by positive regulation of NdhR under enriched CO 2 conditions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: A Salmonella enterica subsp. diarizonae (hereafter S. diarizonae) clinical strain S499 demonstrated unique genomic features. The strain S499 was treated with polymyxin B in vitro to investigate the mechanism of resistance., Methods: S499 was treated with polymyxin B by increasing concentration gradually to obtain a resistant mutant S499V. Whole genomes of the two strains were sequenced using Illumina HiSeq X-10 and PacBio RS II platforms. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to compare the gene expression., Results: The chromosome of strain S499 contained a 40-kb DNA region that was replicated after treatment with polymyxin B and generated a triple tandem DNA repeat region in the chromosome of mutant strain S499V. This repeat region in S499V was flanked by IS1 and contained pmrD, pmrG, and arnBCADTEF operon. In comparison to the homologous 40-kb DNA region of strain S499, a few genes in the repeat DNA region of strain S499V contained truncating mutations that generate two open reading frames (ORFs). The expression of pmrD, pmrG, and arnT was significantly upregulated in S499V., Conclusion: The duplication and overexpression of pmrD, pmrG, and arnT operon may be responsible for the polymyxin B resistance of mutant strain S499V., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Staphylococcus pseudintermedius, which is part of the skin microbiome of dogs, causes a variety of opportunistic infections. These infections may become more difficult to treat due to the formation of biofilm. The capacity of S. pseudintermedius to form biofilm, as well as the associated genes, has not been elucidated. This study evaluated the production and composition of S. pseudintermedius biofilm. Samples were collected from both infected dogs and asymptomatic dogs. Isolates were identified using mass spectrometry and Multiplex-PCR. Biofilm production and composition were assessed using a quantitative microtiter plate assay. The presence of ica operon genes and sps genes was investigated using conventional PCR. The investigation of Agr type and virulence genes was conducted in silico on 24 sequenced samples. All strains could produce strong biofilms, with most of the isolates presenting a polysaccharide biofilm. 63.6% of the isolates carried the complete ica operon (ADBC). All samples showed the presence of the genes spsK, spsA, and spsL, while the distribution of other genes varied. Agr type III was the most prevalent (52.2%). All sequenced samples carried the cytotoxins hlb, luk-S, luk-F, as well as the exfoliative toxins siet and se_int. No isolate displayed other exfoliative toxins. Only LB1733 presented a set of different enterotoxins (sea, seb, sec_canine, seh, sek, sel, and seq). Our findings suggest that S. pseudintermedius is a strong producer of biofilm and carries virulence genes., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Industrial wastewater is a major environmental concern due to its high copper content, which poses significant toxicity to microbial life. Autoinducer-2 (AI-2) can participate in the inter- and intra-species communication and regulate the physiological functions of different bacterial species by producing AI-2 signal molecules. However, there are few research reports on the luxS gene and lsr operon functions for AI-2 in bacteria with a certain tolerance to copper. This study delves into the potential of quorum sensing mechanisms, particularly the AI-2 system, for enhancing microbial resistance to copper toxicity in Klebsiella michiganensis (KM). We detail the critical roles of the luxS gene in AI-2 synthesis and the lsr operon in AI-2 uptake, demonstrating their collective impact on enhancing copper resistance. Our findings show that mutations in the lsr operon, alongside the knockout of the luxS gene in KM strain (KMΔluxSΔlsr), significantly impair the strain's motility (p < 0.0001) and biofilm formation (p < 0.01), underscoring the operon's role in AI-2 transport. These genetic insights are pivotal for developing bioremediation strategies aimed at mitigating copper pollution in wastewater. By elucidating the mechanisms through which KM modulates copper resistance, this study highlights the broader ecological significance of leveraging microbial quorum sensing pathways for sustainable wastewater management., 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.)