Two-component Signaling Systems Research Articles

Translation profiling of stress-induced small proteins reveals a novel link among signaling systems.

Signaling networks allow adaptation to stressful environments by activating genes that counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we identify stress-induced small proteins using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, several of them transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, underscoring their physiological significance in low magnesium stress. Most remarkably, we elucidate an unusual connection via a small membrane protein YoaI, between major signaling networks - PhoR-PhoB and EnvZ-OmpR in E. coli, advancing our understanding of small protein regulators in cellular signaling.

Constitutive activation of two-component systems reveals regulatory network interactions in Streptococcus agalactiae

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

Two-Component Signaling System RegAB Represses Pseudomonas syringae pv. actinidiae T3SS by Directly Binding to the promoter of hrpRS1

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a significant threat to the kiwifruit industry. The two-component signaling systems (TCSs) play a crucial role in regulating the virulence of Pseudomonas syringae (P. syringae), yet their specific function in Psa remains largely unclear. In this study, we found that disrupting the TCS RegAB (encoded by Psa_802/Psa_803) resulted in a notable increase in the pathogenicity of Pseudomonas syringae pv. actinidiae M228 (Psa M228) in host plant and hypersensitive reaction (HR) in nonhost plant. Through comparative transcriptome analysis of the Psa M228 wild-type strain and the regA mutant, we identified the pivotal role of RegA/B in controlling various physiological pathways, including the Type III secretion system (T3SS), a key determinant of Psa virulence. Additionally, we discovered that the RegA does have binding sites in the promoter region of the hrpR/S, and the transcriptional level of the hrpR and other T3SS-related genes increased in the regA deletion strain relative to the Psa M228 wild-type. The DNA-binding affinity of RegA, and therefore the repressor function, is enhanced by its phosphorylation. Our findings unveil the function of TCS RegAB and the regulatory mechanism of T3SS by RegAB in Psa, highlighting the diverse functions of the RegAB system.

FapR regulates HssRS-mediated heme homeostasis in Bacillus anthracis.

Bacillus anthracis, a Gram-positive facultative anaerobe and the causative agent of anthrax, multiplies to extraordinarily high numbers in vertebrate blood, resulting in considerable heme exposure. Heme is an essential nutrient and the preferred iron source for bacteria during vertebrate colonization, but its high redox potential makes it toxic in excess. To regulate heme homeostasis, many Gram-positive bacteria, including B. anthracis, rely on the two-component signaling system HssRS. HssRS comprises the heme sensing histidine kinase HssS, which modulates the activity of the HssR transcription factor to enable bacteria to circumvent heme toxicity. However, the regulation of the HssRS system remains unclear. Here we identify FapR, the transcriptional regulator of fatty acid biosynthesis, as a key factor in HssRS function. FapR plays an important role in maintaining membrane integrity and the localization of the histidine kinase HssS. Specifically, disruption of fapR leads to increased membrane rigidity, which hinders the penetration of HssRS inducers, resulting in the inactivation of HssRS. Furthermore, deletion of fapR affects the loading of HssS onto the cell membrane, compromising its heme sensing function and subsequently reducing endogenous heme biosynthesis. These findings shed light on the molecular mechanisms governing bacterial adaptation to heme stress and provide potential targets for antimicrobial intervention strategies.

Repurposing Hsp90 inhibitors as antimicrobials targeting two-component systems identifies compounds leading to loss of bacterial membrane integrity.

The discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.

Bacterial Histidine Kinase and the Development of Its Inhibitors in the 21st Century.

Bacterial histidine kinase (BHK) is a constituent of the two-component signaling (TCS) pathway, which is responsible for the regulation of a number of processes connected to bacterial pathogenicity, virulence, biofilm development, antibiotic resistance, and bacterial persistence. As BHK regulation is diverse, inhibitors can be developed, such as antibiotic synergists, bacteriostatic/bactericidal agents, virulence inhibitors, and biofilm inhibitors. Inhibition of essential BHK has always been an amenable strategy due to the conserved binding sites of the domains across bacterial species and growth dependence. Hence, an inhibitor of BHK might block multiple TCS regulatory networks. This review describes the TCS system and the role of BHK in bacterial virulence and discusses the available inhibitors of BHK, which is a specific response regulator with essential structural features.

Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator.

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions-Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE's secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of "one gene, one enzyme, one function", which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.

HssS activation by membrane heme defines a paradigm for two-component system signaling in Staphylococcus aureus.

Strict management of intracellular heme pools, which are both toxic and beneficial, is crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme-sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, a heme-binding site is present in the HssS dimer between the membrane and extracellular domains. Heme is embedded in the membrane bilayer with its two protruding porphyrin propionates interacting with two conserved Arg94 and Arg163 that are located extracellularly. Single substitutions of these arginines and two highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic pocket limited the ability of HssS to induce HrtBA synthesis. Combination of the four substitutions abolished HssS activation. Wild-type (WT) HssS copurified with heme from Escherichia coli, whereas heme binding was strongly attenuated in the variants. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation and induce HrtBA-mediated heme extrusion from the membrane. This "gatekeeper" mechanism could limit intracellular diffusion of exogenous heme in S. aureus and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors.IMPORTANCEIn the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity, Staphylococcus aureus expresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphotransfer mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the transmembrane domains (TM) helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen-sensing mechanisms as a prerequisite to the discovery of inhibitors.

Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

Revisiting old friends: updates on the role of two-component signaling systems in Listeria monocytogenes survival and pathogenesis.

Listeria monocytogenes is well recognized for both its broad resistance to stress conditions and its ability to transition from a soil bacterium to an intracellular pathogen of mammalian hosts. The bacterium's impressive ability to adapt to changing environments and conditions requires the rapid sensing of environmental cues and the coordinated response of gene products that enable bacterial growth and survival. Two-component signaling systems (TCSs) have been long recognized for their ability to detect environmental stimuli and transmit those signals into transcriptional responses; however, often the precise nature of the stimulus triggering TCS responses can be challenging to define. L. monocytogenes has up to 16 TCSs that have been recognized based on homology and included in this list are several whose functions remain poorly described. This review highlights the current understanding of the breadth and scope of L. monocytogenes TCS as relates to stress resistance and pathogenesis. Precise signals still often remain elusive, but the gene networks associated with TCSs are providing clues into possible functions.

Bioinformatics analysis of type II toxin-antitoxin systems and regulatory functional assessment of HigBA and SS-ATA in Streptococcus suis1

Toxin-antitoxin (TA) systems, which are prevalent in bacteria and archaea, play diverse roles in bacterial physiology and have been proposed to be significant in stress adaptation. Despite the extensive characterization of numerous TA systems in various bacteria, the investigation of these systems within Streptococcus suis (S. suis) is still limited. Here, we systematically analyzed the type II TA systems of 95 S. suis genomes available in the GenBank database using TAfinder. A total of 612 putative type II TA systems were retrieved and classified into 10 categories by phylogenetic analysis. Notably, an elevated occurrence of these TA systems was observed among the important prevalent serotypes 2, 4, 5, 9, 14, Chz, NCL1, and NCL3 strains. The following study identified the activities of TA systems using two strategies and confirmed the regulatory effect of HigBA on the type VII secretion system in S. suis by measuring β-galactosidase activity and transcriptional changes. Moreover, we unveiled a hitherto uncharacterized, highly prevalent novel TA system, with the composition of antitoxin-toxin-antitoxin (SS-ATA), which regulates the downstream two-component signaling system. Altogether, this study systematically analyzed the type II TA systems within S. suis, highlighting the widespread distribution of HigBA and SS-ATA as important regulatory elements in S. suis.

Mapping niche-specific two-component system requirements in uropathogenic Escherichia coli.

Sensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify-for the first time-a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct.IMPORTANCEWhile two-component system (TCS) signaling has been investigated at depth in model strains of Escherichia coli, there have been no studies to elucidate-at a systems level-which TCSs are important during infection by pathogenic Escherichia coli. Here, we report the generation of a markerless TCS deletion library in a uropathogenic E. coli (UPEC) isolate that can be leveraged for dissecting the role of TCS signaling in different aspects of pathogenesis. We use this library to demonstrate, for the first time in UPEC, that niche-specific colonization is guided by distinct TCS groups.

In silico and structural investigation of sulfonamides targeting VraSR two component system in methicillin-resistant Staphylococcus aureus

Drug-resistant Staphylococcus aureus strains are global health concerns. Several studies have shown that these strains can develop defences against cell wall antibiotics such as β-lactams, glycopeptides and daptomycin which target cell wall biosynthesis. The coordination of these responses have been associated with two component system (TCS) regulated by histidine kinase protein (VraS) and its cognate regulator VraR which influences the target DNA upon signal recognition. Computer-based screening methods, predictions and simulations have emerged as more efficient and quick ways to identify promising new compound leads from large databases against emerging drug targets thus allowing prediction of small select set of molecules for further validations. These combined approaches conserve valuable time and resources. Due to methicillin resistance, sulfonamide-derivative medications have been found to be effective treatment strategy to treat S. aureus infections. The current study used ligand-based virtual screening (LBVS) to identify powerful sulfonamide derivative inhibitors from an antibacterial compound library against VraSR signaling components, VraS and VraR. We identified promising sulfonamide derivative [compound 5: (4-[(1-{[(3,5-Dimethoxyphenyl)Carbamoyl]Methyl}-2,4-Dioxo-1,2,3,4-Tetrahydroquinazolin-3-Yl)Methyl]-N-[(Furan-2-Yl)Methyl]Benzamide)] with reasonable binding parameters of −31.38 kJ/mol and ΔGbind score of −294.32 kJ/mol against ATP binding domain of sensor kinase VraS. We further identified four compounds N1 (PCID83276726), N3 (PCID83276757), N9 (PCID3672584), and N10 (PCID20900589) against VraR DNA binding domain (VraRC) with ΔGbind energies of −190.27, −237.54, −165.21, and −190.88 kJ/mol, respectively. Structural and simulation analyses further suggest their stable interactions with DNA interacting residues and potential to disrupt DNA binding domain dimerization; therefore, it is prudent to further investigate and characterize them as VraR dimer disruptors and inhibit other promoter binding site. Interestingly, the discovery of drugs that target VraS and VraR may open new therapeutic avenues for drug-resistant S. aureus. These predictions based on screening, simulations and binding affinities against VraSR components hold promise for opening novel therapeutic avenues against drug-resistant S. aureus and present opportunities for repositioning efforts. These efforts aim to create analogs with enhanced potency and selectivity against two-component signaling systems that significantly contribute to virulence in MRSA or VRSA. These analyses contribute valuable insights into potential avenues for combating antibiotic-resistant S. aureus through computationally driven drug discovery strategies. Communicated by Ramaswamy H. Sarma

Evolution shapes interaction patterns for epistasis and specific protein binding in a two-component signaling system

The elegant design of protein sequence/structure/function relationships arises from the interaction patterns between amino acid positions. A central question is how evolutionary forces shape the interaction patterns that encode long-range epistasis and binding specificity. Here, we combined family-wide evolutionary analysis of natural homologous sequences and structure-oriented evolution simulation for two-component signaling (TCS) system. The magnitude-frequency relationship of coupling conservation between positions manifests a power-law-like distribution and the positions with highly coupling conservation are sparse but distributed intensely on the binding surfaces and hydrophobic core. The structure-specific interaction pattern involves further optimization of local frustrations at or near the binding surface to adapt the binding partner. The construction of family-wide conserved interaction patterns and structure-specific ones demonstrates that binding specificity is modulated by both direct intermolecular interactions and long-range epistasis across the binding complex. Evolution sculpts the interaction patterns via sequence variations at both family-wide and structure-specific levels for TCS system.

The Caulobacter NtrB-NtrC two-component system bridges nitrogen assimilation and cell development.

A suite of molecular sensory systems enables Caulobacter to control growth, development, and reproduction in response to levels of essential elements. The bacterial enhancer-binding protein (bEBP) NtrC and its cognate sensor histidine kinase, NtrB, are key regulators of nitrogen assimilation in many bacteria, but their roles in Caulobacter metabolism and development are not well defined. Notably, Caulobacter NtrC is an unconventional bEBP that lacks the σ54-interacting loop commonly known as the GAFTGA motif. Here we show that deletion of Caulobacter crescentus ntrC slows cell growth in complex medium and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glutamine synthetase expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring transcription of the glnBA operon, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nutrient limitation. We further identified dozens of direct NtrC-binding sites on the C. crescentus chromosome, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of binding sites align with those of the essential nucleoid-associated protein, GapR, or the cell cycle regulator, MucR1. NtrC is therefore predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter. IMPORTANCE Bacteria balance cellular processes with the availability of nutrients in their environment. The NtrB-NtrC two-component signaling system is responsible for controlling nitrogen assimilation in many bacteria. We have characterized the effect of ntrB and ntrC deletion on Caulobacter growth and development and uncovered a role for spontaneous IS element transposition in the rescue of transcriptional and nutritional deficiencies caused by ntrC mutation. We further defined the regulon of Caulobacter NtrC, a bacterial enhancer-binding protein, and demonstrate that it shares specific binding sites with essential proteins involved in cell cycle regulation and chromosome organization. Our work provides a comprehensive view of transcriptional regulation mediated by a distinctive NtrC protein, establishing its connection to nitrogen assimilation and developmental processes in Caulobacter.

MtrA modulates Mycobacterium tuberculosis cell division in host microenvironments to mediate intrinsic resistance and drug tolerance

The success of Mycobacterium tuberculosis (Mtb) is largely attributed to its ability to physiologically adapt and withstand diverse localized stresses within host microenvironments. Here, we present a data-driven model (EGRIN 2.0) that captures the dynamic interplay of environmental cues and genome-encoded regulatory programs in Mtb. Analysis of EGRIN 2.0 shows how modulation of the MtrAB two-component signaling system tunes Mtb growth in response to related host microenvironmental cues. Disruption of MtrAB by tunable CRISPR interference confirms that the signaling system regulates multiple peptidoglycan hydrolases, among other targets, that are important for cell division. Further, MtrA decreases the effectiveness of antibiotics by mechanisms of both intrinsic resistance and drug tolerance. Together, the model-enabled dissection of complex MtrA regulation highlights its importance as a drug target and illustrates how EGRIN 2.0 facilitates discovery and mechanistic characterization of Mtb adaptation to specific host microenvironments within the host.

Spatiotemporal regulation of the BarA/UvrY two-component signaling system

The BarA/UvrY two-component signal transduction system mediates adaptive responses of Escherichia coli to changes in growth stage. At late exponential growth phase, the BarA sensor kinase autophosphorylates and transphosphorylates UvrY, which activates transcription of the CsrB and CsrC noncoding RNAs. CsrB and CsrC, in turn, sequester and antagonize the RNA binding protein CsrA, which posttranscriptionally regulates translation and/or stability of its target mRNAs. Here, we provide evidence that during stationary phase of growth, the HflKC complex recruits BarA to the poles of the cells and silences its kinase activity. Moreover, we show that during the exponential phase of growth, CsrA inhibits hflK and hflC expression, thereby enabling BarA activation upon encountering its stimulus. Thus, in addition to temporal control of BarA activity, spatial regulation is demonstrated.

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia.

Mycobacterium abscessus (Mab), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for Mab infections are poor due to Mab's inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches Mab must overcome via alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of Mab within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRS Mab , a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of Mycobacterium tuberculosis (Mtb), DosRS Mtb , is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO) in vitro and in vivo. Previously, a small DosR Mab regulon was predicted using bioinformatics based on DosR Mtb motifs however, the role and regulon of DosRS Mab in Mab pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a Mab dosRS knockout strain (MabΔdosRS) to investigate differential gene expression, and phenotype in an in vitro hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate Mab_dosR and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a dosRS mutant strain. Deletion of DosRS Mab led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of Mab to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRS Mab for adaptation of Mab to hypoxia, highlighting a distinct regulon (compared to Mtb) that is significantly larger than previously described, including both genes conserved across mycobacteria as well as Mab-specific genes.

Genomic Island-Encoded Histidine Kinase and Response Regulator Coordinate Mannose Utilization with Virulence in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429-5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429-5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429-5434) involved in mannose utilization. The TCS directly upregulates EDL5429-5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

Transcriptome analysis reveals that yeast extract inhibits synthesis of prodigiosin by Serratia marcescens SDSPY-136

Prodigiosin (2-methyl-3-pentyl-6-methoxyprodiginine) is a valuable medicinal and edible natural pigment derived from Serratia marcescens. How prodigiosin synthesis is suppressed by environmental factors has not been investigated. Previous studies described a low level of prodigiosin production in the presence of yeast extracts. However, we have observed that S. marcescens SDSPY-136 did not synthesize prodigiosin in yeast extract culture. In this study, transcriptome sequencing of yeast extract cultures was used to estimate the metabolic control of the synthetic prodigiosin pathway in S. marcescens. Key phosphorylation enzymes in the glycolysis pathway, 6-phosphofructokinase, and glyceraldehyde 3-phosphate dehydrogenase, were downregulated by yeast extract and other carbon metabolism pathway genes were enhanced. Genes related to ribosomes, amino acid metabolism, and aminoacyl-tRNA biosynthesis were also highly up-regulated. The presence of metal ions in yeast extracts and the accumulation of fermentation metabolites alter the two-component signaling system, which regulated metabolism to various degrees. The results of metal ion testing suggested that prodigiosin inhibition could be caused by metal ions, such as zinc ion. The findings indicate that yeast extract may affect metabolism through multiple pathways in S. marcescens. This research sheds light on the mechanism of prodigiosin regulatory inhibition.