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ABSTRACTST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the “mcsw” morphotype. Two distinct morphotypes (“ntrd” and “msdw”) were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

The adaptation of Salmonella enterica serovar Typhimurium to stress conditions involves expression of genes within the regulon of the alternative sigma factor RpoN (σ54). RpoN-dependent transcription requires an activated bacterial enhancer binding protein (bEBP) that hydrolyzes ATP to remodel the RpoN-holoenzyme-promoter complex for transcription initiation. The bEBP RtcR in S. Typhimurium strain 14028s is activated by genotoxic stress to direct RpoN-dependent expression of the RNA repair operon rsr-yrlBA-rtcBA. The molecular signal for RtcR activation is an oligoribonucleotide with a 3'-terminal 2',3'-cyclic phosphate. We show in S. Typhimurium 14028s that the molecular signal is not a direct product of nucleic acid damage, but signal generation is dependent on a RecA-controlled SOS-response pathway, specifically, induction of prophage Gifsy-1. A genome-wide mutant screen and utilization of Gifsy prophage-cured strains indicated that the nucleoid-associated protein Fis and the Gifsy-1 prophage significantly impact RtcR activation. Directed-deletion analysis and genetic mapping by transduction demonstrated that a three-gene region (STM14_3218-3220) in Gifsy-1, which is variable between S. Typhimurium strains, is required for RtcR activation in strain 14028s and that the absence of STM14_3218-3220 in the Gifsy-1 prophages of S. Typhimurium strains LT2 and 4/74, which renders these strains unable to activate RtcR during genotoxic stress, can be rescued by complementation in cis by the region encompassing STM14_3218-3220. Thus, even though RtcR and the RNA repair operon are highly conserved in Salmonella enterica serovars, RtcR-dependent expression of the RNA repair operon in S. Typhimurium is controlled by a variable region of a prophage present in only some strains. IMPORTANCE The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, RtcA and RtcB, are widely conserved in Proteobacteria. In Salmonella Typhimurium 14028s, genotoxic stress activates RtcR to direct RpoN-dependent expression of the rsr-yrlBA-rtcBA operon. This work identifies key elements of a RecA-dependent pathway that generates the signal for RtcR activation in strain 14028s. This signaling pathway requires the presence of a specific region within the prophage Gifsy-1, yet this region is absent in most other wild-type Salmonella strains. Thus, we show that the activity of a widely conserved regulatory protein can be controlled by prophages with narrow phylogenetic distributions. This work highlights an underappreciated phenomenon where bacterial physiological functions are altered due to genetic rearrangement of prophages.

The Gram‐negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high‐throughput transposon sequencing (Tn‐seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN‐deficient mutant strain was defective in tracheal colonization when co‐inoculated with the wild‐type strain. rpoN encodes σ54, a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions. [ABSTRACT FROM AUTHOR]

Vibrio alginolyticus is a prevalent pathogen in both humans and marine species, exhibiting high adaptability to various adverse environmental conditions. Our previous studies have shown that ΔrpoN formed three enhanced biofilm types, including spectacular surface-attached biofilm (SB), scattered pellicle biofilm (PB), and colony rugosity. However, the precise mechanism through which rpoN regulates biofilm formation has remained unclear. Based on the critical role of Vibrio exopolysaccharide (VPS) in biofilm formation, several genes related to the production and regulation of VPS were characterized in V. alginolyticus. Our findings from mutant strains indicated that VPS has complete control over the formation of rugose colony morphology and PB, while it only partially contributes to SB formation. Among the four transcriptional regulators of the vps gene cluster, vpsR and VA3545 act as promoters, whereas VA3546 and VA2703 function as repressors. Through transcriptome analysis and c-di-GMP concentration determination, VA0356 and VA3580 which encoded diguanylate cyclase were found to mediate the ΔrpoN biofilm formation. As a central regulator, rpoN governed biofilm formation through two regulatory pathways. Firstly, it directly bound to the upstream region of VA4206 to regulate the expression of the vps gene cluster (VA4206-VA4196). Secondly, it directly and indirectly modulated c-di-GMP synthesis gene VA3580 and VA0356, respectively, thereby affecting c-di-GMP concentration and subsequently influencing the expression of vps transcription activators vpsR and VA3545. Under conditions promoting SB formation, ΔrpoN was unable to thrive below the liquid level due to significantly reduced activities of three catalytic enzymes (ACK, ADH, and ALDH) involved in pyruvate metabolism, but tended to reproduce in air-liquid interface, a high oxygen niche compared to the liquid phase. In conclusion, both exopolysaccharide synthesis and oxygen-related metabolism contributed to ΔrpoN biofilm formation. The role of RpoN-mediated hypoxic metabolism and biofilm formation were crucial for comprehending the colonization and pathogenicity of V. alginolyticus in hosts, providing a novel target for treating V. alginolyticus in aquatic environments and hosts.

Genome-scale studies of the bacterial regulatory network have been leveraged by declining sequencing cost and advances in ChIP (chromatin immunoprecipitation) methods. Of which, ChIP-exo has proven competent with its near-single base-pair resolution. While several algorithms and programs have been developed for different analytical steps in ChIP-exo data processing, there is a lack of effort in incorporating them into a convenient bioinformatics pipeline that is intuitive and publicly available. In this paper, we developed ChIP-exo Analysis Pipeline (ChEAP) that executes the one-step process, starting from trimming and aligning raw sequencing reads to visualization of ChIP-exo results. The pipeline was implemented on the interactive web-based Python development environment – Jupyter Notebook, which is compatible with the Google Colab cloud platform to facilitate the sharing of codes and collaboration among researchers. Additionally, users could exploit the free GPU and CPU resources allocated by Colab to carry out computing tasks regardless of the performance of their local machines. The utility of ChEAP was demonstrated with the ChIP-exo datasets of RpoN sigma factor in E. coli K-12 MG1655. To analyze two raw data files, ChEAP runtime was 2 min and 25 s. Subsequent analyses identified 113 RpoN binding sites showing a conserved RpoN binding pattern in the motif search. ChEAP application in ChIP-exo data analysis is extensive and flexible for the parallel processing of data from various organisms.

Abstract Background RpoN, also known as σ54, first reported in Escherichia coli, is a subunit of RNA polymerase that strictly controls the expression of different genes by identifying specific promoter elements. RpoN has an important regulatory function in carbon and nitrogen metabolism and participates in the regulation of flagellar synthesis, bacterial motility and virulence. However, little is known about the effect of RpoN in Plesiomonas shigelloides. Results To identify pathways controlled by RpoN, RNA sequencing (RNA-Seq) of the WT and the rpoN deletion strain was carried out for comparison. The RNA-seq results showed that RpoN regulates ~ 13.2% of the P. shigelloides transcriptome, involves amino acid transport and metabolism, glycerophospholipid metabolism, pantothenate and CoA biosynthesis, ribosome biosynthesis, flagellar assembly and bacterial secretion system. Furthermore, we verified the results of RNA-seq using quantitative real-time reverse transcription PCR, which indicated that the absence of rpoN caused downregulation of more than half of the polar and lateral flagella genes in P. shigelloides, and the ΔrpoN mutant was also non-motile and lacked flagella. In the present study, the ability of the ΔrpoN mutant to kill E. coli MG1655 was reduced by 54.6% compared with that of the WT, which was consistent with results in RNA-seq, which showed that the type II secretion system (T2SS-2) genes and the type VI secretion system (T6SS) genes were repressed. By contrast, the expression of type III secretion system genes was largely unchanged in the ΔrpoN mutant transcriptome and the ability of the ΔrpoN mutant to infect Caco-2 cells was also not significantly different compared with the WT. Conclusions We showed that RpoN is required for the motility and contributes to the killing ability of P. shigelloides and positively regulates the T6SS and T2SS-2 genes.

ABSTRACT The functional significance of rpoN genes that encode two sigma factors in the Bradyrhizobium sp. strain DOA9 has been reported to affect colony formation, root nodulation characteristics, and symbiotic interactions with Aeschynomene americana. rpoN mutant strains are defective in cellular surface polysaccharide (CSP) production compared with the wild-type (WT) strain, and they accordingly exhibit smaller colonies and diminished symbiotic effectiveness. To gain deeper insights into the changes in CSP composition and the nodules of rpoN mutants, we employed synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy. FTIR analysis of the CSP revealed the absence of specific components in the rpoN mutants, including lipids, carboxylic groups, polysaccharide-pyranose rings, and β-galactopyranosyl residues. Nodules formed by DOA9WT exhibited a uniform distribution of lipids, proteins, and carbohydrates; mutant strains, particularly DOA9∆rpoNp:ΩrpoNc, exhibited decreased distribution uniformity and a lower concentration of C=O groups. Furthermore, Fe K-edge X-ray absorption near-edge structure and extended X-ray absorption fine structure analyses revealed deficiencies in the nitrogenase enzyme in the nodules of DOA9∆rpoNc and DOA9∆rpoNp:ΩrpoNc mutants; nodules from DOA9WT and DOA9∆rpoNp exhibited both leghemoglobin and the nitrogenase enzyme. IMPORTANCE This work provides valuable insights into how two rpoN genes affect the composition of cellular surface polysaccharides (CSPs) in Bradyrhizobium sp., which subsequently dictates root nodule chemical characteristics and nitrogenase production. We used advanced synchrotron methods, including synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy (XAS), for the first time in this field to analyze CSP components and reveal the biochemical changes occurring within nodules. These cutting-edge techniques confer significant advantages by providing detailed molecular information, enabling the identification of specific functional groups, chemical bonds, and biomolecule changes. This research not only contributes to our understanding of plant-microbe interactions but also establishes a foundation for future investigations and potential applications in this field. The combined use of the synchrotron-based FTIR and XAS techniques represents a significant advancement in facilitating a comprehensive exploration of bacterial CSPs and their implications in plant-microbe interactions.

ABSTRACT Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA299–356, an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by ΔompA299–356. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA299–356 mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJΔOmpA299–356. KJΔOmpA299–356 complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJΔOmpA299–356. Next, the involvement of the three σ factors in the ΔompA299–356-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the ΔompA299–356-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σE decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the ΔompA299–356-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation. IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal β-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σP and σE expression levels. The outcomes of σP and σE activation are different and are linked to β-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.

The genetic basis underlying loss‐of‐virulence mutations that arise among natural phytopathogen populations is not well documented. In this study, we examined the virulence of 377 isolates of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) that were isolated from 76 kiwifruit orchards suffering from bacterial canker disease. Eighty‐four nonpathogenic isolates were identified in 40 orchards. A nonpathogenic isolate G166 was found to be defective in hrpL transcription and the downstream type III secretion system (T3SS)‐dependent phenotypes. Comparative genomics and complementary expression assay revealed that a single‐base "G" insertion in the hrpL promoter blocks gene transcription by reducing promoter activity. The electrophoretic mobility shift assay showed that the genetic variation impairs σ54/promoter binding during gene transcription under hrp‐inducing conditions, resulting in lower expression of hrpL. A PCR‐restriction fragment length polymorphism assay was performed to trace the evolutionary history of this mutation, which revealed the independent onset of genetic variations in natural Psa3 populations. We also found that nonpathogenic variants outperformed virulent Psa3 bacteria for both epiphytic and apoplast colonization of kiwifruit leaves in mixed inoculations. Our study highlights a novel mechanism for loss of virulence in Psa3 and provides insight into bacterial adaptive evolution under natural settings. [ABSTRACT FROM AUTHOR]

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium's ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during freeliving and symbiotic states. [ABSTRACT FROM AUTHOR]

Erwinia amylovora causes a devastating fire blight disease in apples and pears. One of the main virulence determinants in E. amylovora is the hypersensitive response (HR) and pathogenicity (hrp)-type III secretion system (T3SS), which is activated by the RpoN-HrpL sigma factor cascade. However, the RpoN regulon in E. amylovora has not been investigated. In this study, we determined the RpoN regulon in E. amylovora by combining RNA-seq transcriptomic analysis with in silico binding site analysis. RNA-seq revealed that 262 genes, approximately 7.5% genes in the genome of E. amylovora, were differentially transcribed in the rpoN mutant as compared with the wild type. Specifically, genes associated with virulence, motility, nitrogen assimilation, the PspF system, stress response, and arginine biosynthesis are positively regulated by RpoN, whereas genes associated with biosynthesis of amino acids and sorbitol transport are negatively regulated by RpoN. In silico binding site analysis identified 46 potential target genes with a putative RpoN binding site, and the upstream sequences of six, three, and three genes also contain putative GlnG, PspF, and YfhA binding sites, respectively. Overall, RpoN directly regulates genes associated with virulence, nitrogen assimilation, the PspF system, motility and the YfhA/YfhK two-component regulatory system. [ABSTRACT FROM AUTHOR]

Pseudomonas protegens is an important biocontrol agent with the ability to suppress plant pathogens and promote plant growth. P. protegens' ability to endure hyperosmotic stress is crucial to its effectiveness as a biocontrol agent. This study elucidated potassium's role and mechanism of action in enabling the hyperosmotic tolerance of P. protegens. Potassium was observed to significantly improve the growth of P. protegens under hyperosmotic conditions. Four functionally redundant potassium transporters, KdpA1, KdpA2, TrkH, and Kup, were identified in P. protegens , of which KdpA2 and TrkH were particularly important for its growth under hyperosmotic conditions. Potassium enhanced the biofilm formation and cell membrane stability of P. protegens under hyperosmotic conditions. In addition, we revealed that K+ stimulates the expression of several genes related to DNA damage repair in P. protegens under hyperosmotic conditions. Further experiments revealed that the DNA repair-related recG induced by potassium contributes to P. protegens ' hyperosmotic tolerance. We also found that the sigma factor RpoN participates in the hyperosmotic adaptation of P. protegens. Furthermore, we revealed that the opuCABCD operon, whose expression is induced by potassium through RpoN, serves as the key pathway through which betaine, choline, and carnitine improve the hyperosmotic tolerance of P. protegens. [ABSTRACT FROM AUTHOR]

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium’s ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.

Vibrio parahaemolyticus is a foodborne pathogen that can colonize the small intestine of the host and cause diarrhea. The alternative sigma factor RpoN plays a vital role in regulating motility, carbon utilization and affects host colonization in V. parahaemolyticus RIMD2210633. In this study, transcriptome and phenotypic analysis further expanded our understanding of the RpoN regulon in V. parahaemolyticus. A deletion mutant of rpoN (ΔrpoN) was subjected to RNA-seq for systemic identification of the RpoN-controlled genes. Compared with the wild-type (WT), 399 genes were differentially expressed in the ΔrpoN strain. Moreover, 264 genes were down-regulated in the ΔrpoN strain, including those associated with nitrogen utilization (VP0118), glutamine synthetase (VP0121), formate dehydrogenase (VP1511 and VP1513-VP1515), quorum sensing (opaR and luxZ), polar flagellar systems, and type VI secretion system 2 (T6SS2). Quantitative real-time reverse transcription PCR (qRT-PCR) and electrophoretic mobility shift assay (EMSA) further confirmed that RpoN could directly bind to the promoters of these genes associated with polar flagellar systems (flgB and fliE), lateral flagellar systems (flgB2 and lafA), T6SS2 (hcp2 and VPA1044) and glutamine synthetase (VP0121), and then positively regulate the expression of these systems. A RpoN-binding motif was identified in V. parahaemolyticus using the MEME suite and verified by the EMSA. Besides, the deletion of rpoN caused a significant decrease in hemolytic activity, adhesion, and cytotoxicity. Our results provide new cues to better understand the regulatory networks of RpoN protein to motility, T6SS2, and metabolism in V. parahaemolyticus. [ABSTRACT FROM AUTHOR]

Background: RpoN, also known as σ54, first reported in Escherichia coli, is a subunit of RNA polymerase that strictly controls the expression of different genes by identifying specific promoter elements. RpoN has an important regulatory function in carbon and nitrogen metabolism and participates in the regulation of flagellar synthesis, bacterial motility and virulence. However, little is known about the effect of RpoN in Plesiomonas shigelloides. Results: To identify pathways controlled by RpoN, RNA sequencing (RNA-Seq) of the WT and the rpoN deletion strain was carried out for comparison. The RNA-seq results showed that RpoN regulates ~ 13.2% of the P. shigelloides transcriptome, involves amino acid transport and metabolism, glycerophospholipid metabolism, pantothenate and CoA biosynthesis, ribosome biosynthesis, flagellar assembly and bacterial secretion system. Furthermore, we verified the results of RNA-seq using quantitative real-time reverse transcription PCR, which indicated that the absence of rpoN caused downregulation of more than half of the polar and lateral flagella genes in P. shigelloides, and the ΔrpoN mutant was also non-motile and lacked flagella. In the present study, the ability of the ΔrpoN mutant to kill E. coli MG1655 was reduced by 54.6% compared with that of the WT, which was consistent with results in RNA-seq, which showed that the type II secretion system (T2SS-2) genes and the type VI secretion system (T6SS) genes were repressed. By contrast, the expression of type III secretion system genes was largely unchanged in the ΔrpoN mutant transcriptome and the ability of the ΔrpoN mutant to infect Caco-2 cells was also not significantly different compared with the WT. Conclusions: We showed that RpoN is required for the motility and contributes to the killing ability of P. shigelloides and positively regulates the T6SS and T2SS-2 genes. [ABSTRACT FROM AUTHOR]

The variable sigma (σ) subunit of the bacterial RNA polymerase (RNAP) holoenzyme, which is responsible for promoter specificity and open complex formation, plays a strategic role in the response to environmental changes. Salmonella enterica serovar Typhimurium utilizes the housekeeping σ70 and five alternative sigma factors, including σ54 The σ54-RNAP differs from other σ-RNAP holoenzymes in that it forms a stable closed complex with the promoter and requires ATP hydrolysis by an activated cognate bacterial enhancer binding protein (bEBP) to transition to an open complex and initiate transcription. In S. Typhimurium, σ54-dependent promoters normally respond to one of 13 different bEBPs, each of which is activated under a specific growth condition. Here, we utilized a constitutively active, promiscuous bEBP to perform a genome-wide identification of σ54-RNAP DNA binding sites and the transcriptome of the σ54 regulon of S. Typhimurium. The position and context of many of the identified σ54 RNAP DNA binding sites suggest regulatory roles for σ54-RNAP that connect the σ54 regulon to regulons of other σ factors to provide a dynamic response to rapidly changing environmental conditions.IMPORTANCE The alternative sigma factor σ54 (RpoN) is required for expression of genes involved in processes with significance in agriculture, bioenergy production, bioremediation, and host-microbe interactions. The characterization of the σ54 regulon of the versatile pathogen S. Typhimurium has expanded our understanding of the scope of the σ54 regulon and how it links to other σ regulons within the complex regulatory network for gene expression in bacteria.

The extracellular matrix is essential for the biofilm formation of food spoilers. Pseudomonas fluorescens PF07 is a previous isolate from spoiled marine fish; however, the genes involved in the extracellular matrix formation of PF07 biofilms remain poorly defined. In this study, PF07 formed a wrinkled macrocolony biofilm through the high production of extracellular matrix. The genes involved in biofilm matrix formation and regulation were screened and identified by RNA-seq-dependent transcriptomic analysis and gene knock-out analysis. The macrocolony biofilms of PF07 grown for 5 days (PF07_5d) were compared with those grown for 1 day (PF07_1d). A total of 1,403 genes were significantly differentially expressed during biofilm formation. These mainly include the genes related to biofilm matrix proteins, polysaccharides, rhamnolipids, secretion system, biofilm regulation, and metabolism. Among them, functional amyloid genes fapABCDE were highly upregulated in the mature biofilm, and the operon fapA-E had a –24/–12 promoter dependent on the sigma factor RpoN. Moreover, the RNA-seq analyses of the rpoN mutant, compared with PF07, revealed 159 genes were differentially expressed in the macrocolony biofilms, and fapA-E genes were positively regulated by RpoN. In addition, the deletion mutants of fapC , rpoN , and brfA (a novel gene coding for an RpoN-dependent transcriptional regulator) were defective in forming mature macrocolony biofilms, solid surface-associated (SSA) biofilms, and pellicles, and they showed significantly reduced biofilm matrices. The fap genes were significantly downregulated in Δ brfA , as in Δ rpoN. These findings suggest that the functional amyloid Fap is the main component of PF07 biofilm matrices, and RpoN may directly regulate the transcription of fap genes, in conjunction with BrfA. These genes may serve as potential molecular targets for screening new anti-biofilm agents or for biofilm detection in food environments. [ABSTRACT FROM AUTHOR]

The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen Clostridioides difficile. To explore the contribution of SigL to C. difficile biology, we generated sigL -disruption mutants (sigL::erm) in strains belonging to two phylogenetically distinct lineages—the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleiotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 sigL::erm relative to the isogenic parent strain but remained unchanged in BI-1 sigL::erm. In contrast, secreted toxin levels were significantly elevated only in the BI-1 sigL::erm mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences C. difficile 's virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes. [ABSTRACT FROM AUTHOR]

Abstract Background Glutamate and aspartate are preferred nutrients for a variety of microorganisms. In the case for many Pseudomonas spp., utilization of these amino acids is believed to be dependent on a transporter complex comprised of a periplasmic-solute binding protein (AatJ), two permease domains (AatQM) and an ATP-binding component (AatP). Notably, expression of this transporter complex is hypothesized to be regulated at the transcriptional level by the enhancer-binding protein AauR and the alternative sigma factor RpoN. The purpose of the current study was to determine the biological significance of the putative aatJ-aatQMP operon and its regulatory aauR and rpoN genes in the utilization of L-glutamate, L-glutamine, L-aspartate and L-asparagine in Pseudomonas aeruginosa PAO1. Results Deletion of the aatJ-aatQMP, aauR or rpoN genes did not affect the growth of P. aeruginosa PAO1 on L-glutamate, L-glutamine, L-aspartate and L-asparagine equally. Instead, only growth on L-glutamate as the sole carbon source was abolished with the deletion of any one of these genes. Interestingly, growth of the aauR mutant on L-glutamate was readily restored via plasmid-based expression of the aatQMP genes, suggesting that it is the function of AatQMP (and not AatJ) that is limiting in the absence of the aauR gene. Subsequent analysis of beta-galactosidase reporters revealed that both aatJ and aatQ were induced in response to L-glutamate, L-glutamine, L-aspartate or L-asparagine in a manner dependent on the aauR and rpoN genes. In addition, both aatJ and aatQ were expressed at reduced levels in the absence of the inducing-amino acids and the regulatory aauR and rpoN genes. The expression of the aatJ-aatQMP genes is, therefore, multifaceted. Lastly, the expression levels of aatJ were significantly higher (> 5 fold) than that of aatQ under all tested conditions. Conclusions The primary function of AauR in P. aeruginosa PAO1 is to activate expression of the aatJ-aatQMP genes in response to exogenous acidic amino acids and their amide derivatives. Importantly, it is the AauR-RpoN mediated induction of the aatQMP genes that is the pivotal factor enabling P. aeruginosa PAO1 to effectively utilize or consume L-glutamate as a sole or preferred nutrient.

The extracellular matrix is essential for the biofilm formation of food spoilers. Pseudomonas fluorescens PF07 is a previous isolate from spoiled marine fish; however, the genes involved in the extracellular matrix formation of PF07 biofilms remain poorly defined. In this study, PF07 formed a wrinkled macrocolony biofilm through the high production of extracellular matrix. The genes involved in biofilm matrix formation and regulation were screened and identified by RNA-seq-dependent transcriptomic analysis and gene knock-out analysis. The macrocolony biofilms of PF07 grown for 5 days (PF07_5d) were compared with those grown for 1 day (PF07_1d). A total of 1,403 genes were significantly differentially expressed during biofilm formation. These mainly include the genes related to biofilm matrix proteins, polysaccharides, rhamnolipids, secretion system, biofilm regulation, and metabolism. Among them, functional amyloid genes fapABCDE were highly upregulated in the mature biofilm, and the operon fapA-E had a –24/–12 promoter dependent on the sigma factor RpoN. Moreover, the RNA-seq analyses of the rpoN mutant, compared with PF07, revealed 159 genes were differentially expressed in the macrocolony biofilms, and fapA-E genes were positively regulated by RpoN. In addition, the deletion mutants of fapC, rpoN, and brfA (a novel gene coding for an RpoN-dependent transcriptional regulator) were defective in forming mature macrocolony biofilms, solid surface-associated (SSA) biofilms, and pellicles, and they showed significantly reduced biofilm matrices. The fap genes were significantly downregulated in ΔbrfA, as in ΔrpoN. These findings suggest that the functional amyloid Fap is the main component of PF07 biofilm matrices, and RpoN may directly regulate the transcription of fap genes, in conjunction with BrfA. These genes may serve as potential molecular targets for screening new anti-biofilm agents or for biofilm detection in food environments.

The alternative sigma factor SigL (Sigma-54) facilitates bacterial adaptation to the extracellular environment by modulating the expression of defined gene subsets. A homolog of the gene encoding SigL is conserved in the diarrheagenic pathogen Clostridioides difficile. To explore the contribution of SigL to C. difficile biology, we generated sigL-disruption mutants (sigL::erm) in strains belonging to two phylogenetically distinct lineages—the human-relevant Ribotype 027 (strain BI-1) and the veterinary-relevant Ribotype 078 (strain CDC1). Comparative proteomics analyses of mutants and isogenic parental strains revealed lineage-specific SigL regulons. Concomitantly, loss of SigL resulted in pleiotropic and distinct phenotypic alterations in the two strains. Sporulation kinetics, biofilm formation, and cell surface-associated phenotypes were altered in CDC1 sigL::erm relative to the isogenic parent strain but remained unchanged in BI-1 sigL::erm. In contrast, secreted toxin levels were significantly elevated only in the BI-1 sigL::erm mutant relative to its isogenic parent. We also engineered SigL overexpressing strains and observed enhanced biofilm formation in the CDC1 background, and reduced spore titers as well as dampened sporulation kinetics in both strains. Thus, we contend that SigL is a key, pleiotropic regulator that dynamically influences C. difficile's virulence factor landscape, and thereby, its interactions with host tissues and co-resident microbes.

The stringent response mediated by the signal molecule (p)ppGpp is involved in response to multiple environmental stresses and control of various physiological processes. Studies have revealed that (p)ppGpp strongly affects the formation and maintenance of several bacterial biofilms. However, the specific regulatory roles of (p)ppGpp in biofilms, especially in the expression of genes related to cell motility and exopolysaccharides (EPSs) production, remain poorly understood. We recently reported two (p)ppGpp synthetase genes relA and spoT from the epizootic pathogen Vibrio alginolyticus. Herein, we found that the (p)ppGpp synthetase genes of V. alginolyticus contributed to biofilm formation at low cell density and biofilm detachment at high cell density, respectively, in polystyrene microtiter plates. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that the expression levels of both EPSs and motility associated genes were consistent with the development of biofilms. Besides, the (p)ppGpp synthetase gene spoT was found to be closely involved in the regulation of flagellum, smooth/translucent colony morphology and spotty pellicle at the air-liquid interface. Interestingly, pleiotropic phenotypes of Δ relA Δ spoT were similar to that of the rpoN (σ54) deletion mutant. Meanwhile, the absence of (p)ppGpp synthetase genes significantly reduced the expression levels of rpoN at low cell density, suggesting that (p)ppGpp may mediate the formation via positively affecting the alternative sigma factor RpoN. These findings allow us to propose (p)ppGpp as a crucial regulator for biofilm development in V. alginolyticus , in view of the regulatory roles of relA and spoT in cell motility and EPSs production. [ABSTRACT FROM AUTHOR]

Abstract Carbapenem-resistant P. aeruginosa (CRPA) has become a serious public health problem and the biofilm formation aggravates this problem. The study aimed to evaluate the occurrence of β-lactamases and quorum sensing (QS) genes in CRPA isolates, analyze production of biofilm, evaluate the response against meropenem (MPM) and∕or polymyxin B (POL B) and its association with azythromicin (AZT) using quantum dots (QDs) and proteomic analysis. Six CRPA isolates were analyzed. β-lactamases and QS genes were search using specific PCRs and were tested for biofilm production by quantitative technique. A CRPA isolate, containing blaKPC gene and biofilm-producing, was selected to assess its response to therapy using QDs and the MALDI-TOF. The β-lactamase detected was blaKPC in 66.7% of the isolates. All isolates were biofilm producers and carriers of the QS genes. QDs-MPM conjugates triggered the formation of biofilm and the association with AZT inhibited this effect. Proteomics analysis showed that treatments with MPM or POL B suppressed the expression of the transglycosylase protein, while combined therapy with AZT induced expression of the RpoN protein. Thus, this study shows that the use of fluorescence combined with the proteomics analysis was promising to understand how a CRPA strain reacts to antimicrobial treatment.

ABSTRACT Bacterial chemotaxis affords motile bacteria the ability to navigate the environment to locate niches for growth and survival. At the molecular level, chemotaxis depends on chemoreceptor signaling arrays that interact with cytoplasmic proteins to control the direction of movement. In Azospirillum brasilense, chemotaxis is mediated by two distinct chemotaxis pathways: Che1 and Che4. Both Che1 and Che4 are critical in the A. brasilense free-living and plant-associated lifestyles. Here, we use whole-cell proteomics and metabolomics to characterize the role of chemotaxis in A. brasilense physiology. We found that mutants lacking CheA1 or CheA4 or both are affected in nonchemotaxis functions, including major changes in transcription, signaling transport, and cell metabolism. We identify specific effects of CheA1 and CheA4 on nitrogen metabolism, including nitrate assimilation and nitrogen fixation, that may depend, at least, on the transcriptional control of rpoN, which encodes RpoN, a global regulator of metabolism, including nitrogen. Consistent with proteomics, the abundance of several nitrogenous compounds (purines, pyrimidines, and amino acids) changed in the metabolomes of the chemotaxis mutants relative to the parental strain. Further, we uncover novel, and yet uncharacterized, layers of transcriptional and posttranscriptional control of nitrogen metabolism regulators. Together, our data reveal roles for CheA1 and CheA4 in linking chemotaxis and nitrogen metabolism, likely through control of global regulatory networks. IMPORTANCE Bacterial chemotaxis is widespread in bacteria, increasing competitiveness in diverse environments and mediating associations with eukaryotic hosts ranging from commensal to beneficial and pathogenic. In most bacteria, chemotaxis signaling is tightly linked to energy metabolism, with this coupling occurring through the sensory input of several energy-sensing chemoreceptors. Here, we show that in A. brasilense the chemotaxis proteins have key roles in modulating nitrogen metabolism, including nitrate assimilation and nitrogen fixation, through novel and yet unknown regulations. These results are significant given that A. brasilense is a model bacterium for plant growth promotion and free-living nitrogen fixation and is used as a bio-inoculant for cereal crops. Chemotaxis signaling in A. brasilense thus links locomotor behaviors to nitrogen metabolism, allowing cells to continuously and reciprocally adjust metabolism and chemotaxis signaling as they navigate gradients.

Background: Glutamate and aspartate are preferred nutrients for a variety of microorganisms. In the case for many Pseudomonas spp., utilization of these amino acids is believed to be dependent on a transporter complex comprised of a periplasmic-solute binding protein (AatJ), two permease domains (AatQM) and an ATP-binding component (AatP). Notably, expression of this transporter complex is hypothesized to be regulated at the transcriptional level by the enhancer-binding protein AauR and the alternative sigma factor RpoN. The purpose of the current study was to determine the biological significance of the putative aatJ-aatQMP operon and its regulatory aauR and rpoN genes in the utilization of L-glutamate, L-glutamine, L-aspartate and L-asparagine in Pseudomonas aeruginosa PAO1. Results: Deletion of the aatJ-aatQMP, aauR or rpoN genes did not affect the growth of P. aeruginosa PAO1 on L-glutamate, L-glutamine, L-aspartate and L-asparagine equally. Instead, only growth on L-glutamate as the sole carbon source was abolished with the deletion of any one of these genes. Interestingly, growth of the aauR mutant on L-glutamate was readily restored via plasmid-based expression of the aatQMP genes, suggesting that it is the function of AatQMP (and not AatJ) that is limiting in the absence of the aauR gene. Subsequent analysis of beta-galactosidase reporters revealed that both aatJ and aatQ were induced in response to L-glutamate, L-glutamine, L-aspartate or L-asparagine in a manner dependent on the aauR and rpoN genes. In addition, both aatJ and aatQ were expressed at reduced levels in the absence of the inducing-amino acids and the regulatory aauR and rpoN genes. The expression of the aatJ-aatQMP genes is, therefore, multifaceted. Lastly, the expression levels of aatJ were significantly higher (> 5 fold) than that of aatQ under all tested conditions. Conclusions: The primary function of AauR in P. aeruginosa PAO1 is to activate expression of the aatJ-aatQMP genes in response to exogenous acidic amino acids and their amide derivatives. Importantly, it is the AauR-RpoN mediated induction of the aatQMP genes that is the pivotal factor enabling P. aeruginosa PAO1 to effectively utilize or consume L-glutamate as a sole or preferred nutrient. [ABSTRACT FROM AUTHOR]

ABSTRACT RpoN, an alternative sigma factor commonly known as σ54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN binding motif located at 103 inter- and intragenic sites on both sense and antisense strands. Deletion of rpoN had a large impact on gene expression, including downregulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the antisense strand commonly reduced expression of genes carried by the corresponding sense strand. IMPORTANCE The alternative sigma factor RpoN (σ54), which is widely distributed in eubacteria, has been implicated in controlling gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection, and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis, and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first study of its kind of RpoN in Y. pseudotuberculosis, revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA, providing important information about RpoN regulation in enterobacteria.

Evolution experiments in the laboratory have focused heavily on model organisms, often to the exclusion of clinically relevant pathogens. The foodborne bacterial pathogen Campylobacter jejuni belongs to a genus whose genomes are small compared to those of its closest genomic relative, the free-living genus Sulfurospirillum, suggesting genome reduction during the course of evolution to host association. In an in vitro experiment, C. jejuni serially passaged in rich medium in the laboratory exhibited loss of flagellar motility–an essential function for host colonization. At early time points the motility defect was often reversible, but after 35 days of serial culture, motility was irreversibly lost in most cells in 5 independently evolved populations. Population re-sequencing revealed disruptive mutations to genes in the flagellar transcriptional cascade, rpoN (σ54)—therefore disrupting the expression of the genes σ54 regulates—coupled with deletion of rpoN in all evolved lines. Additional mutations were detected in virulence-related loci. In separate in vivo experiments, we demonstrate that a phase variable (reversible) motility mutant carrying an adenine deletion within a homopolymeric tract resulting in truncation of the flagellar biosynthesis gene fliR was deficient for colonization in a C57BL/6 IL-10–/– mouse disease model. Re-insertion of an adenine residue partially restored motility and ability to colonize mice. Thus, a pathogenic C. jejuni strain was rapidly attenuated by experimental laboratory evolution and demonstrated genomic instability during this evolutionary process. The changes observed suggest C. jejuni is able to evolve in a novel environment through genome reduction as well as transition, transversion, and slip-strand mutations.

Recurrent painful ophthalmoplegic neuropathy (RPON) is a very rare disease characterized by recurrent attacks (at least two) of unilateral headache associated with ipsilateral ophthalmoplegia due to paresis of one or more cranial motor nerves, not due to any orbital, parasellar, or posterior fossa lesions. The differential diagnoses for this condition are broad. In addition to disability during an acute attack, this disease could also cause a permanent neurologic deficit. The understanding of RPON pathogenesis has changed over time, leading to a change in the classification of this disorder between editions of the International Classification of Headache Disorders, in which the condition was moved from the chapter on migraine to the chapter on cranial neuralgias and central causes of facial pain. There is no consensus on the pathogenesis of RPON. It is possible that multiple pathogenic mechanisms underlie various clinical forms of the disease. A depiction of pathologic analyses of patients with radiologically confirmed changes in the affected nerves during and outside of attacks would significantly contribute to knowledge of its pathogenesis. Brain imaging should be performed in each patient during an acute RPON attack and at a regular schedule between attacks. Further case reports and case series are required before further conclusions can be made regarding RPON pathogenesis and proposals for treatment options. [ABSTRACT FROM AUTHOR]

Labrenzia aggregata LZB033 (Rhodobacteraceae), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a large number of transcriptional regulators which may be important for its adaptation to diverse marine environments. The alternative 54 factor (RpoN) is a central regulator of many bacteria, regulating the transcription of multiple genes and controlling important cellular functions. However, the exact role of RpoN in Labrenzia spp. is unknown. In this study, an in-frame rpoN deletion mutant was constructed in LZB033, and the function of RpoN was determined. To systematically identify RpoN-controlled genes, we performed a detailed analysis of gene expression differences between the wild-type strain and the ΔrpoN mutant using RNA sequencing. The expression of 175 genes was shown to be controlled by RpoN. Subsequent phenotypic assays showed that the ΔrpoN mutant was attenuated in flagellar biosynthesis and swimming motility, utilized up to 13 carbon substrates differently, lacked the ability to assimilate malic acid, and displayed markedly decreased biofilm formation. In addition, stress response assays showed that the ΔrpoN mutant was impaired in the ability to survive under different challenge conditions, including osmotic stress, oxidative stress, temperature changes, and acid stress. Moreover, both the DMSP synthesis and catabolism rates of LZB033 decreased after rpoN was knocked out. Our work provides essential insight into the regulatory function of RpoN, revealing that RpoN is a key determinant for LZB033 flagellar formation, motility, biofilm formation, and environmental fitness, as well as DMSP production and degradation. [ABSTRACT FROM AUTHOR]

The sigma factor 54 (σ54) controls the expression of many genes in response to nutritional and environmental conditions. There are two σ54 genes, rpoN1 (XAC1969) and rpoN2 (XAC2972), in Xanthomonas citri subsp. citri. To investigate their functions, the deletion mutants ΔrpoN1, ΔrpoN2 and ΔrpoN1N2 were constructed in this study. All the mutants delayed canker development in low concentration inoculation in citrus plants. The bacterial growth of mutants was retarded in the medium supplemented with nitrogen and carbon resources. Under either condition, the influence degree caused by deletion of rpoN2 was larger than the deletion of rpoN1. Remarkably, the mutant ΔrpoN1 showed a reduction in cell motility, while the mutant ΔrpoN2 increased cell motility. Our data suggested that the rpoN1 and rpoN2 play diverse roles in X. citri subsp. citri.

The rhizobacterium Pseudomonas protegens H78 biosynthesizes a number of antibiotic compounds, including pyoluteorin, 2,4-diacetylphloroglucinol, and pyrrolnitrin. Here, we investigated the global regulatory function of the nitrogen metabolism-related sigma factor RpoN in P. protegens H78 through RNA-seq and phenotypic analysis. During the mid- to late-log growth phase, transcriptomic profiling revealed that 562 genes were significantly upregulated, and 502 genes were downregulated by at least twofold at the RNA level in the rpoN deletion mutant in comparison with the wild-type strain H78. With respect to antibiotics, Plt biosynthesis and the expression of its operon were positively regulated, while Prn biosynthesis and the expression of its operon were negatively regulated by RpoN. RpoN is responsible for the global activation of operons involved in flagellar biogenesis and assembly, biofilm formation, and bacterial mobility. In contrast, RpoN was shown to negatively control a number of secretion system operons including one type VI secretion system operon (H1-T6SS), two pilus biogenesis operons (Flp/Tad-T4b pili and Csu-T1 pili), and one polysaccharide biosynthetic operon (psl). In addition, two operons that are involved in mannitol and inositol utilization are under the positive regulation of RpoN. Consistent with this result, the ability of H78 to utilize mannitol or inositol as a sole carbon source is positively influenced by RpoN. Taken together, the RpoN-mediated global regulation is mainly involved in flagellar biogenesis and assembly, bacterial mobility, biofilm formation, antibiotic biosynthesis, secretion systems, and carbon utilization in P. protegens H78. [ABSTRACT FROM AUTHOR]

Abstract: Based on the analysis of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and mass spectrometry, flagellin FliC and a key member of the type VI secretion system Hcp1 were found to be secreted from Pseudomonas putida KT2442 cells in the late exponential phase, but in the early exponential phase only FliC was secreted. Relevant genes fleQ and rpoN were then individually deleted in P. putida KT2442, and the expression and transcription of fliC and hcp1 in these mutant strains were analyzed. In both △fleQ and △rpoN mutant cells, Hcp1 but not FliC was secreted in the late exponential phase, and it was secreted even in the early exponential phase. Transcriptomic analysis of △fleQ cells shows that most of the genes relevant to the type VI secretion system, including hcp1, were upregulated; several genes involved in 3′,5′‐cyclic diguanylic acid (c‐di‐GMP) synthesis and degradation were also significantly regulated. To further study the regulation process of the type VI secretion system, the phosphodiesterase BifA was overexpressed to lower the intracellular c‐di‐GMP level. The results demonstrate that FleQ positively regulates flagella biosynthesis but negatively regulates the type VI secretion system in P. putida. This finding is important for understanding the function and regulation of the type VI secretion system. [ABSTRACT FROM AUTHOR]

RpoN (or σ54) is the key sigma factor for the regulation of transcription of nitrogen fixation genes in diazotrophic bacteria, which include α- and β-rhizobia. Our previous studies showed that an rpoN mutant of the β-rhizobial strain Paraburkholderia phymatum STM815T formed root nodules on Phaseolus vulgaris cv. Negro jamapa, which were unable to reduce atmospheric nitrogen into ammonia. In an effort to further characterize the RpoN regulon of P. phymatum, transcriptomics was combined with a powerful metabolomics approach. The metabolome of P. vulgaris root nodules infected by a P. phymatum rpoN Fix- mutant revealed statistically significant metabolic changes compared to wild-type Fix+ nodules, including reduced amounts of chorismate and elevated levels of flavonoids. A transcriptome analysis on Fix- and Fix+ nodules--combined with a search for RpoN binding sequences in promoter regions of regulated genes--confirmed the expected control of σ54 on nitrogen fixation genes in nodules. The transcriptomic data also allowed us to identify additional target genes, whose differential expression was able to explain the observed metabolite changes in numerous cases. Moreover, the genes encoding the two-component regulatory system NtrBC were downregulated in root nodules induced by the rpoN mutant, and contained a putative RpoN binding motif in their promoter region, suggesting direct regulation. The construction and characterization of an ntrB mutant strain revealed impaired nitrogen assimilation in free-living conditions, as well as a noticeable symbiotic phenotype, as fewer but heavier nodules were formed on P. vulgaris roots. [ABSTRACT FROM AUTHOR]

Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated twitching motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoNOH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoNOH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoNOH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4Pmediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoNOH11, pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoNOH11 plays a key role for RpoNOH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated twitching motility and biofilm formation in L. enzymogenes OH11. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa has developed diverse strategies to respond and adapt to antibiotic stress. Among the factors that modulate survival in the presence of antibiotics, alternative sigma factors play an important role. Here, we demonstrate that the alternative sigma factor RpoN (σ54) promotes survival in the presence of tobramycin. The tobramycin-sensitive phenotype of logarithmic phase ΔrpoN mutant cells is suppressed by the loss of the alternative sigma factor RpoS. Transcriptional analysis indicated that RpoN positively regulates the expression of RsmA, an RNA-binding protein, in the P. aeruginosa stationary growth phase in a nutrient-rich medium. The loss of RpoS led to the upregulation of gacA expression in the nutrient-limited medium-grown stationary phase cells. Conversely, in the logarithmic growth phase, the ΔrpoS mutant demonstrated lower expression of gacA, underscoring a regulatory role of RpoS for GacA. Supplementation of tobramycin to stationary phase ΔrpoN mutant cells grown in nutrient-rich medium resulted in decreased expression of gacA, relA, and rpoS without altering the expression of rsmA relative to wild-type PAO1. The observed downregulation of gacA and relA in the ΔrpoN mutant in the presence of tobramycin could be reversed through the mutation of rpoS in the ΔrpoN mutant background. The tobramycin-tolerant phenotype of the ΔrpoNΔrpoS mutant logarithmic phase cells may be associated with the expression of relA, which remained unresponsive upon addition of tobramycin. The logarithmic phase ΔrpoS and ΔrpoNΔrpoS mutant cells demonstrated increased expression of gacA in response to tobramycin. Together, these results suggest that a complex regulatory interaction between RpoN, RpoS, the Gac/Rsm pathway, and RelA modulates the P. aeruginosa response to tobramycin.

Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans. In this pathway, EbpA, a σ54 activator and putative prokaryotic enhancerbinding protein (EBP), and the alternative sigma factor RpoN (σ54) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with L-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans. [ABSTRACT FROM AUTHOR]