Colistin resistance mutations in phoQ can sensitize Klebsiella pneumoniae to IgM-mediated complement killing.

Due to multi-drug resistance, physicians increasingly use the last-resort antibiotic colistin to treat infections with the Gram-negative bacterium Klebsiella pneumoniae. Unfortunately, K. pneumoniae can also develop colistin resistance. Interestingly, colistin resistance has dual effects on bacterial clearance by the immune system. While it increases resistance to antimicrobial peptides, colistin resistance has been reported to sensitize certain bacteria for killing by human serum. Here we investigate the mechanisms underlying this increased serum sensitivity, focusing on human complement which kills Gram-negatives via membrane attack complex (MAC) pores. Using in vitro evolved colistin resistant strains and a fluorescent MAC-mediated permeabilization assay, we showed that two of the three tested colistin resistant strains, Kp209_CSTR and Kp257_CSTR, were sensitized to MAC. Transcriptomic and mechanistic analyses focusing on Kp209_CSTR revealed that a mutation in the phoQ gene locked PhoQ in an active state, making Kp209_CSTR colistin resistant and MAC sensitive. Detailed immunological assays showed that complement activation on Kp209_CSTR in human serum required specific IgM antibodies that bound Kp209_CSTR but did not recognize the wild-type strain. Together, our results show that developing colistin resistance affected recognition of Kp209_CSTR and its killing by the immune system.

Hypermucoviscosity Regulator RmpD Interacts with Wzc and Controls Capsular Polysaccharide Chain Length.

Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.

Novel Drivers of Virulence in Clostridioides difficile Identified via Context-Specific Metabolic Network Analysis.

The pathogen Clostridioides difficile causes toxin-mediated diarrhea and is the leading cause of hospital-acquired infection in the United States. Due to growing antibiotic resistance and recurrent infection, targeting C. difficile metabolism presents a new approach to combat this infection. Genome-scale metabolic network reconstructions (GENREs) have been used to identify therapeutic targets and uncover properties that determine cellular behaviors. Thus, we constructed C. difficile GENREs for a hypervirulent isolate (strain [str.] R20291) and a historic strain (str. 630), validating both with in vitro and in vivo data sets. Growth simulations revealed significant correlations with measured carbon source usage (positive predictive value [PPV] ≥ 92.7%), and single-gene deletion analysis showed >89.0% accuracy. Next, we utilized each GENRE to identify metabolic drivers of both sporulation and biofilm formation. Through contextualization of each model using transcriptomes generated from in vitro and infection conditions, we discovered reliance on the pentose phosphate pathway as well as increased usage of cytidine and N-acetylneuraminate when virulence expression is reduced, which was subsequently supported experimentally. Our results highlight the ability of GENREs to identify novel metabolite signals in higher-order phenotypes like bacterial pathogenesis. IMPORTANCE Clostridioides difficile has become the leading single cause of hospital-acquired infections. Numerous studies have demonstrated the importance of specific metabolic pathways in aspects of C. difficile pathophysiology, from initial colonization to regulation of virulence factors. In the past, genome-scale metabolic network reconstruction (GENRE) analysis of bacteria has enabled systematic investigation of the genetic and metabolic properties that contribute to downstream virulence phenotypes. With this in mind, we generated and extensively curated C. difficile GENREs for both a well-studied laboratory strain (str. 630) and a more recently characterized hypervirulent isolate (str. R20291). In silico validation of both GENREs revealed high degrees of agreement with experimental gene essentiality and carbon source utilization data sets. Subsequent exploration of context-specific metabolism during both in vitro growth and infection revealed consistent patterns of metabolism which corresponded with experimentally measured increases in virulence factor expression. Our results support that differential C. difficile virulence is associated with distinct metabolic programs related to use of carbon sources and provide a platform for identification of novel therapeutic targets.

The Small Protein RmpD Drives Hypermucoviscosity in Klebsiella pneumoniae.

Klebsiella pneumoniae has a remarkable ability to cause a wide range of human diseases. It is divided into two broad classes: classical strains that are a notable problem in health care settings due to multidrug resistance, and hypervirulent (hv) strains that are historically drug sensitive but able to establish disease in immunocompetent hosts. Alarmingly, there has been an increased frequency of clinical isolates that have both drug resistance and hv-associated genes. One such gene, rmpA, encodes a transcriptional regulator required for maximal capsule (cps) gene expression and confers hypermucoviscosity (HMV). This link has resulted in the assumption that HMV is caused by elevated capsule production. However, we recently reported a new cps regulator, RmpC, and ΔrmpC mutants have reduced cps expression but retain HMV, suggesting that capsule production and HMV may be separable traits. Here, we report the identification of a small protein, RmpD, that is essential for HMV but does not impact capsule. RmpD is 58 residues with a putative N-terminal transmembrane domain and highly positively charged C-terminal half, and it is conserved among other hv K. pneumoniae strains. Expression of rmpD in trans complements both ΔrmpD and ΔrmpA mutants for HMV, suggesting that RmpD is the key driver of this phenotype. The rmpD gene is located between rmpA and rmpC, within an operon regulated by RmpA. These data, combined with our previous work, suggest a model in which the RmpA-associated phenotypes are largely due to RmpA activating the expression of rmpD to produce HMV and rmpC to stimulate cps expression.IMPORTANCE Capsule is a critical virulence factor in Klebsiella pneumoniae, in both antibiotic-resistant classical strains and hypervirulent strains. Hypervirulent strains usually have a hypermucoviscosity (HMV) phenotype that contributes to their heightened virulence capacity, but the production of HMV is not understood. The transcriptional regulator RmpA is required for HMV and also activates capsule gene expression, leading to the assumption that HMV is caused by hyperproduction of capsule. We have identified a new gene (rmpD) required for HMV but not for capsule production. This distinction between HMV and capsule production will promote a better understanding of the mechanisms of hypervirulence, which is in great need given the alarming increase in clinical isolates with both drug resistance and hypervirulence traits.

The intersection of capsule gene expression, hypermucoviscosity and hypervirulence in Klebsiella pneumoniae.

For ∼30 years, two distinct groups of clinical isolates of Klebsiella pneumoniae have been recognized. Classical strains (cKp) are typically isolated from patients with some degree of immunocompromise and are not virulent in mouse models of infection whereas hypervirulent strains (hvKp) are associated with community acquired invasive infections and are highly virulent in mouse models of infection. Hyperproduction of capsule and a hypermucoviscous colony phenotype have been strongly associated with the hypervirulence of hvKp strains. Recent studies have begun to elucidate the relationship between capsule gene expression, hypermucoviscosity and hypervirulence. Additionally, genes associated with hyperproduction of capsule and hypermucoviscosity in hvKp strains have been identified in a few cKp isolates. However, it is not clear how the acquisition of these genes impacts the virulence of cKp isolates. A better understanding of the potential risks of these strains is particularly important given that many of them are resistant to multiple antibiotics, including carbapenems.

A Klebsiella pneumoniae Regulatory Mutant Has Reduced Capsule Expression but Retains Hypermucoviscosity.

The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA The ΔrmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains.IMPORTANCEKlebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

Identification of Two Regulators of Virulence That Are Conserved in Klebsiella pneumoniae Classical and Hypervirulent Strains.

Klebsiella pneumoniae is widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity of K. pneumoniae isolates complicates our ability to understand the virulence of K. pneumoniae Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors. Klebsiella encodes numerous MarR-like proteins, and they likely contribute to the ability of K. pneumoniae to respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all the marR homologues in a murine pneumonia model and found that two (kvrA and kvrB) significantly impacted the virulence of K1 and K2 capsule type hypervirulent (hv) strains and that kvrA affected the virulence of a sequence type 258 (ST258) classical strain. In the hv strains, kvrA and kvrB mutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription from galF and manC promoters that drive expression of capsule synthesis genes. In contrast, kvrA and kvrB mutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical and hv strains but the effect on virulence may not be exclusively due to effects on capsule production.IMPORTANCE In addition to having a reputation as the causative agent for hospital-acquired infections as well as community-acquired invasive infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. Due to the rapid emergence of carbapenem resistance among K. pneumoniae strains, a better understanding of virulence mechanisms and identification of new potential drug targets are needed. This study identified two novel regulators (KvrA and KvrB) of virulence in K. pneumoniae and demonstrated that their effect on virulence in invasive strains is likely due in part to effects on capsule production (a major virulence determinant) and hypermucoviscosity. KvrA also impacts the virulence of classical strains but does not appear to affect capsule gene expression in this strain. KvrA and KvrB are conserved among K. pneumoniae strains and thus could regulate capsule expression and virulence in diverse strains regardless of capsule type.

A Serendipitous Mutation Reveals the Severe Virulence Defect of a Klebsiella pneumoniae fepB Mutant.

Klebsiella pneumoniae is considered a significant public health threat because of the emergence of multidrug-resistant strains and the challenge associated with treating life-threatening infections. Capsule, siderophores, and adhesins have been implicated as virulence determinants of K. pneumoniae, yet we lack a clear understanding of how this pathogen causes disease. In a previous screen for virulence genes, we identified a potential new virulence locus and constructed a mutant (smr) with this locus deleted. In this study, we characterize the smr mutant and show that this mutation renders K. pneumoniae avirulent in a pneumonia model of infection. The smr mutant was expected to have a deletion of three genes, but subsequent genome sequencing indicated that a much larger deletion had occurred. Further analysis of the deleted region indicated that the virulence defect of the smr mutant could be attributed to the loss of FepB, a periplasmic protein required for import of the siderophore enterobactin. Interestingly, a ΔfepB mutant was more attenuated than a mutant unable to synthesize enterobactin, suggesting that additional processes are affected. As FepB is highly conserved among the members of the family Enterobacteriaceae, therapeutic targeting of FepB may be useful for the treatment of Klebsiella and other bacterial infections. IMPORTANCE In addition to having a reputation as the causative agent of several types of hospital-acquired infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of infections, including urinary tract infections, pneumonia, and sepsis. Because of the rapid emergence of carbapenem resistance among Klebsiella strains, there is a dire need for a better understanding of virulence mechanisms and identification of new drug targets. Here, we identify the periplasmic transporter FepB as one such potential target.

The YsrS Paralog DygS Has the Capacity To Activate Expression of the Yersinia enterocolitica Ysa Type III Secretion System.

UNLABELLED: The Yersinia enterocolitica Ysa type III secretion system (T3SS) is associated with intracellular survival, and, like other characterized T3SSs, it is tightly controlled. Expression of the ysa genes is only detected following growth at low temperatures (26°C) and in high concentrations of sodium chloride (290 mM) in the medium. The YsrSTR phosphorelay (PR) system is required for ysa expression and likely responds to NaCl. During our investigations into the Ysr PR system, we discovered that genes YE3578 and YE3579 are remarkably similar to ysrR and ysrS, respectively, and are probably a consequence of a gene duplication event. The amino acid differences between YE3578 and ysrR are primarily clustered into two short regions. The differences between YE3579 and ysrS are nearly all located in the periplasmic sensing domain; the cytoplasmic domains are 98% identical. We investigated whether these paralogs were capable of activating ysa gene expression. We found that the sensor paralog, named DygS, is capable of compensating for loss of ysrS, but the response regulator paralog, DygR, cannot complement a ysrR gene deletion. In addition, YsrR, but not DygR, interacts with the histidine phosphorelay protein YsrT. Thus, DygS likely activates ysa gene expression in response to a signal other than NaCl and provides an example of a phosphorelay system in which two sensor kinases feed into the same regulatory pathway. IMPORTANCE: All organisms need mechanisms to promote survival in changing environments. Prokaryotic phosphorelay systems are minimally comprised of a histidine kinase (HK) that senses an extracellular stimulus and a response regulator (RR) but can contain three or more proteins. Through gene duplication, a unique hybrid HK was created. We show that, while the hybrid appears to retain all of the phosphorelay functions, it responds to a different signal than the original. Both HKs transmit the signal to the same RR, which activates a promoter that transcribes a set of genes encoding a type III secretion system (T3SS) whose function is not yet evident. The significance of this work lies in finding that two HKs regulate this T3SS, highlighting its importance.

Childhood toxic stress: a community role in health promotion for occupational therapists.

People who experience the toxic stress of recurrent traumatic events in childhood have a higher risk for mental and physical health problems throughout life. Occupational therapy practitioners have a remarkable opportunity to be involved in addressing this significant public health problem. As health care practitioners already situated in the community, we have a responsibility to lead and assist in establishing and implementing occupation-based programs and to nurture the links between the child welfare system and existing intervention systems. In this article, we review the current research on toxic stress and recommendations made by other health care disciplines and offer strategies for occupational therapy practitioners to begin a dialogue on this critical, emerging issue.

A phenotype at last: essential role for the Yersinia enterocolitica Ysa type III secretion system in a Drosophila melanogaster S2 cell model.

The highly pathogenic Yersinia enterocolitica strains have a chromosomally encoded type III secretion system (T3SS) that is expressed and functional in vitro only when the bacteria are cultured at 26 °C. Mutations that render this system nonfunctional are slightly attenuated in the mouse model of infection only following an oral inoculation and only at early time points postinfection. The discrepancy between the temperature required for the Ysa gene expression and the physiological temperature required for mammalian model systems has made defining the role of this T3SS challenging. Therefore, we explored the use of Drosophila S2 cells as a model system for studying Ysa function. We show here that Y. enterocolitica is capable of infecting S2 cells and replicating intracellularly to high levels, an unusual feature of this pathogen. Importantly, we show that the Ysa T3SS is required for robust intracellular replication. A secretion-deficient mutant lacking the secretin gene, ysaC, is defective in replication within S2 cells, marking the first demonstration of a pronounced Ysa-dependent virulence phenotype. Establishment of S2 cells as a model for Y. enterocolitica infection provides a versatile tool to elucidate the role of the Ysa T3SS in the life cycle of this gastrointestinal pathogen.

Development of a community mobility skills course for people who use mobility devices.

OBJECTIVE: We assessed mobility device skills in a lived-in environment on a community mobility skills course (CMSC) and related those skills to previously demonstrated skills in a controlled environment on an inside mobility skills course (IMSC). METHOD: Six mobility device users were selected from 91 adults who had previously completed the IMSC. Each of the following device groups was represented: power wheelchair, manual wheelchair, scooter, cane, crutch, and walker. CMSC tasks were modified from IMSC tasks and designed using materials in the community. RESULTS: All participants completed at least half of the CMSC tasks faster than they completed the corresponding IMSC tasks. The number of tasks on which participants improved and the amount of improvement varied by difficulty of task and mobility device used. CONCLUSION: Some mobility device skills used in controlled environments appear to transfer to community settings; others do not. Skills required for community participation may partially depend on mobility device used.

Identification of YsrT and evidence that YsrRST constitute a unique phosphorelay system in Yersinia enterocolitica.

Two-component systems (TCS) and phosphorelay systems are mechanisms used by bacteria and fungi to quickly adapt to environmental changes to produce proteins necessary for survival in new environments. Bacterial pathogens use TCS and phosphorelay systems to regulate genes necessary to establish infection within their hosts, including type III secretion systems (T3SS). The Yersinia enterocolitica ysa T3SS is activated in response to NaCl by YsrS and YsrR, a putative hybrid sensor kinase and a response regulator, respectively. Hybrid TCS consist of a sensor kinase that typically has three well-conserved sites of phosphorylation: autophosphorylation site H1, D1 within a receiver domain, and H2 in the histidine phosphotransferase (HPt) domain. From H2, the phosphoryl group is transferred to D2 on the response regulator. A curious feature of YsrS is that it lacks the terminal HPt domain. We report here the identification of the HPt-containing protein (YsrT) that provides this activity for the Ysr system. YsrT is an 82-residue protein predicted to be cytosolic and α-helical in nature and is encoded by a gene adjacent to ysrS. To demonstrate predicted functions of YsrRST as a phosphorelay system, we introduced alanine substitutions at H1, D1, H2, and D2 and tested the mutant proteins for the ability to activate a ysaE-lacZ reporter. As expected, substitutions at H1, H2, and D2 resulted in a loss of activation of ysaE expression. This indicates an interruption of normal protein function, most likely from loss of phosphorylation. A similar result was expected for D1; however, an intriguing "constitutive on" phenotype was observed. In addition, the unusual feature of a separate HPt domain led us to compare the sequences surrounding the ysrS-ysrT junction in several Yersinia strains. In every strain examined, ysrT is a separate gene, leading to speculation that there is a functional advantage to YsrT being an independent protein.

Synchronous gene expression of the Yersinia enterocolitica Ysa type III secretion system and its effectors.

Type III secretion systems (T3SSs) are complex units that consist of many proteins. Often the proteins are encoded as a cohesive unit on virulence plasmids, but several systems have their various components dispersed around the chromosome. The Yersinia enterocolitica Ysa T3SS is such a system, where the apparatus genes, some regulatory genes, and four genes encoding secreted proteins (ysp genes) are contained in a single locus. The remaining ysp genes and at least one additional regulator are found elsewhere on the chromosome. Expression of ysa genes requires conditions of high ionic strength, neutral/basic pH, and low temperatures (26 degrees C) and is stimulated by exposure to solid surfaces. The AraC-like regulator YsaE and the dual-function chaperone/regulator SycB are required to stimulate the sycB promoter, which transcribes sycB and probably yspBCDA as well. The putative phosphorelay proteins YsrRS (located at the distal end of the ysa locus) and RcsB, the response regulator of the RcsBCD phosphorelay system, are required to initiate transcription at the ysaE promoter, which drives transcription of many apparatus genes. In this work, we sought to determine which ysp genes were coordinately regulated with the genes within the ysa locus. We found that six unlinked ysp genes responded to NaCl and required YsaE/SycB, YsrRS, and RcsB for expression. Three ysp genes had unique patterns, one of which was unaffected by all elements tested except NaCl. Thus, while the ysp genes were likely to have been acquired independently, most have acquired a synchronous regulatory pattern.

YspM, a newly identified Ysa type III secreted protein of Yersinia enterocolitica.

Yersinia enterocolitica has three type three secretion systems, the flagellar, the plasmid Ysc type III secretion system (T3SS), and the chromosomal Ysa T3SS. The Ysc T3SS, through the proteins it secretes (Yops), prevents phagocytosis of Y. enterocolitica and is required for disease processes in the mouse host. Recent data demonstrate a role for the Ysa T3SS during initial colonization of the mouse via secretion of Ysps (Yersinia secreted proteins). This work characterizes the discovery of a newly identified Ysa type III secreted protein, YspM. Expression of yspM is regulated by temperature, NaCl concentration, and other known regulators of the ysa system. In addition, YspM is translocated into host cells via the Ysa T3SS. YspM is homologous to proteins classified as GDSL bacterial lipases, which possess a catalytic triad of amino acids (Ser, Asp, and His) located in three of five blocks of amino acid identity. Sequence analysis of the JB580v strain of Y. enterocolitica shows that, due to a premature stop codon, it no longer encodes the fifth block of amino acid identity containing the predicted catalytic histidine. However, seven other biotype 1B strains sequenced did possess the domain. A functional difference between the forms was revealed when YspM was expressed in Saccharomyces cerevisiae. Yeast growth was uninhibited when YspM from JB580v was expressed but greatly inhibited when YspM from Y295 (YspM(Y295)) was expressed. Site-directed mutagenesis of the histidine of YspM(Y295) ablated the toxic effects. These results indicate that YspM is secreted by the Ysa T3SS and that, possibly due to lipase activity, it targets eukaryotic cellular component(s).

Environmental stimuli affecting expression of the Ysa type three secretion locus.

Yersinia enterocolitica has two type III secretion systems (TTSS): The well characterized Ysc-Yop system and the relatively uncharacterized Ysa-Ysp system. Detection of Ysps in culture supernatants has only been observed in vitro when cultures are grown at low temperature (26 degrees C) and in high salt (290mM NaCl). Previous reports demonstrated that expression from the ysaE promoter was activated by high salt. In this study, we report a new environmental stimulus for ysa gene expression; in the presence of high salt, growth on solid surface stimulates expression 7-fold compared to growth in high salt broth. These new data indicate that, in the presence of salt, solid surface is an extremely robust signal for the Ysa system.

The Escherichia coli Fis promoter is regulated by changes in the levels of its transcription initiation nucleotide CTP.

Expression of the Escherichia coli nucleoid-associated protein Fis (factor for inversion stimulation) is controlled at the transcriptional level in accordance with the nutritional availability. It is highly expressed during early logarithmic growth phase in cells growing in rich medium but poorly expressed in late logarithmic and stationary phase. However, fis mRNA expression is prolonged at high levels throughout the logarithmic and early stationary phase when the preferred transcription initiation site (+1C) is replaced with A or G, indicating that initiation with CTP is a required component of the regulation pattern. We show that RNA polymerase-fis promoter complexes are short lived and that transcription is stimulated over 20-fold from linear or supercoiled DNA if CTP is present during formation of initiation complexes, which serves to stabilize these complexes. Use of fis promoter fusions to lacZ indicated that fis promoter transcription is sensitive to the intracellular pool of the predominant initiating NTP. Growth conditions resulting in increases in CTP pools also result in corresponding increases in fis mRNA levels. Measurements of NTP pools performed throughout the growth of the bacterial culture in rich medium revealed a dramatic increase in all four NTP levels during the transition from stationary to logarithmic growth phase, followed by reproducible oscillations in their levels during logarithmic growth, which later decrease during the transition from logarithmic to stationary phase. In particular, CTP pools fluctuate in a manner consistent with a role in regulating fis expression. These observations support a model whereby fis expression is subject to regulation by the availability of its initiating NTP.

Regulation of the Ysa type III secretion system of Yersinia enterocolitica by YsaE/SycB and YsrS/YsrR.

Yersinia enterocolitica biovar 1B contains two type III secretion systems (TTSSs), the plasmid-encoded Ysc-Yop system and the chromosomally encoded Ysa-Ysp system. Proteins secreted from the Ysa TTSS (Ysps) have only been detected in vitro when cells are cultured at 26 degrees C in a high-NaCl medium. However, the exact role of the Ysa TTSS is unclear. Thus, investigations into the regulation of this system may help elucidate the role of the Ysps during the life cycle of Y. enterocolitica. Here we present evidence that the AraC-like regulator YsaE acts together with the chaperone SycB to regulate transcription of the sycByspBCDA operon, a phenomenon similar to that seen in the closely related Salmonella SPI-1 and Shigella flexneri Mxi-Spa-Ipa TTSSs. Deletion of either sycB or ysaE results in a twofold reduction in the activity of a sycB-lacZ fusion compared to the wild type. In a reconstituted Escherichia coli system, transcription of sycB was activated sixfold only when both YsaE and SycB were present, demonstrating that they are necessary for activation. ysrR and ysrS are located near the ysa genes and encode a putative two-component regulatory system. Mutations in either gene indicated that both YsrR and YsrS were required for secretion of Ysps. In addition, transcription from sycB-lacZ and ysaE-lacZ fusions was decreased 6.5- and 25-fold, respectively, in the ysrS mutant compared to the wild type. Furthermore, in the absence of NaCl, the activity of ysaE-lacZ was reduced 25-fold in the wild-type and DeltaysrS strains, indicating that YsrS is probably required for the salt-dependent expression of the ysa locus. These results suggest that the putative two-component system YsrRS may be a key element in the regulatory cascade for the Ysa TTSS.

Factors affecting start site selection at the Escherichia coli fis promoter.

Transcription initiation with CTP is an uncommon feature among Escherichia coli sigma(70) promoters. The fis promoter (fis P), which is subject to growth phase-dependent regulation, is among the few that predominantly initiate transcription with CTP. Mutations in this promoter that cause a switch from utilization of CTP to either ATP or GTP as the initiation nucleotide drastically alter its growth phase regulation pattern, suggesting that the choice of the primary initiating nucleotide can significantly affect its regulation. To better understand what factors influence this choice in fis P, we made use of a series of promoter mutations that altered the nucleotide or position used for initiation. Examination of these promoters indicates that start site selection is determined by a combination of factors that include preference for a nucleotide distance from the -10 region (8 > 7 > 9 >> 6 >> 10 > 11), initiation nucleotide preference (A = G >> CTP > or = UTP), the DNA sequence surrounding the initiation region, the position of the -35 region, and changes in the intracellular nucleoside triphosphate pools. We describe the effects that each of these factors has on start site selection in the fis P and discuss the interplay between position and nucleotide preference in this important process.