Jun-Rong Wei

The LuxR family protein SpnR functions as a negative regulator of N‐acylhomoserine lactone‐dependent quorum sensing in Serratia marcescens

Serratia marcescens SS‐1 produces at least four N ‐acylhomoserine lactones (AHLs) which were identified using high‐resolution mass spectrometry and chemical synthesis, as N‐ (3‐oxohexanoyl) homo‐serine lactone (3‐oxo‐C6‐HSL), N ‐hexanoyl‐ (C6‐HSL), N ‐heptanoyl (C7‐HSL) and N ‐octanoyl‐ (C8‐HSL) homoserine lactone. These AHLs are synthesized via the LuxI homologue SpnI, and regulate via the LuxR homologue SpnR, the production of the red pigment, prodigiosin, the nuclease, NucA, and a biosurfactant which facilitates surface translocation. spnR overexpression and spnR gene deletion show that SpnR, in contrast to most LuxR homologues, acts as a negative regulator. spnI overexpression, the provision of exogenous AHLs and spnI gene deletion suggest that SpnR is de‐repressed by 3‐oxo‐C6‐HSL. In addition, long chain AHLs antagonize the biosurfactant‐mediated surface translocation of S. marcescens SS‐1. Upstream of spnI there is a gene which we have termed spnT . spnI and spnT form an operon and although database searches failed to reveal any spnT homologues, overexpression of this novel gene negatively affected both sliding motility and prodigiosin production.

Depletion of antibiotic targets has widely varying effects on growth

It is often assumed that antibiotics act on the most vulnerable cellular targets, particularly those that require limited inhibition to block growth. To evaluate this assumption, we developed a genetic method that can inducibly deplete targeted proteins and that mimics their chemical inactivation. We applied this system to current antibiotic targets in mycobacteria. Although depleting some antibiotic targets significantly perturbs bacterial growth, surprisingly, we found that reducing the levels of other targets by more than 97% had little or no effect on growth. For one of these targets, dihydrofolate reductase, metabolic analysis suggested that depletion mimics the use of subinhibitory concentrations of the antibiotic trimethroprim. These observations indicate that some drug targets can exist at levels much higher than are needed to support growth. However, protein depletion can be used to identify promising drug targets that are particularly vulnerable to inhibition.

A Phosphorylated Pseudokinase Complex Controls Cell Wall Synthesis in Mycobacteria

Structure-function studies in mycobacteria reveal how a Ser-Thr protein kinase and pseudokinase work together to regulate the synthesis of the bacterial cell wall. The Bacterial Cell Wall Construction Foremen The bacterial peptidoglycan cell wall is essential for viability and pathogenesis and represents the target of many antibacterial drugs. In Mycobacterium tuberculosis, which causes tuberculosis, the transmembrane protein MviN is required for peptidoglycan synthesis and contains a kinase-like domain not found in the orthologous proteins of other bacteria. Structural analysis by Gee et al. revealed that, although the kinase homology domain adopted a conserved kinase fold, the protein was an inactive pseudokinase. Biochemical analysis showed that this pseudokinase was a substrate for the Ser-Thr kinase PknB, which is activated by peptidoglycan fragments. Structural and biochemical analysis revealed a high-affinity interaction between the FHA domain–containing protein FhaA and phosphorylated MviN. Conditional depletion or overexpression experiments in vivo suggested that PknB-mediated phosphorylation of the pseudokinase domain of MviN enabled the inhibition of MviN by FhaA. Thus, this protein kinase–pseudokinase–FHA cascade appears to serve as a homeostatic regulator of cell wall metabolism. Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks.

The RssAB Two-Component Signal Transduction System in Serratia marcescens Regulates Swarming Motility and Cell Envelope Architecture in Response to Exogenous Saturated Fatty Acids

Serratia marcescens swarms at 30 degrees C but not at 37 degrees C on a nutrient-rich (LB) agar surface. Mini-Tn5 mutagenesis of S. marcescens CH-1 yielded a mutant (WC100) that swarms not only vigorously at 37 degrees C but also earlier and faster than the parent strain swarms at 30 degrees C. Analysis of this mutant revealed that the transposon was inserted into a gene (rssA) predicted to encode a bacterial two-component signal transduction sensor kinase, upstream of which a potential response regulator gene (rssB) was located. rssA and rssB insertion-deletion mutants were constructed through homologous recombination, and the two mutants exhibited similar swarming phenotypes on LB swarming agar, in which swarming not only occurred at 37 degrees C but also initiated at a lower cell density, on a surface with a higher agar concentration, and more rapidly than the swarming of the parent strain at 30 degrees C. Both mutants also exhibited increased hemolysin activity and altered cell surface topologies compared with the parent CH-1 strain. Temperature and certain saturated fatty acids (SFAs) were found to negatively regulate S. marcescens swarming via the action of RssA-RssB. Analysis of the fatty acid profiles of the parent and the rssA and rssB mutants grown at 30 degrees C or 37 degrees C and under different nutrition conditions revealed a relationship between cellular fatty acid composition and swarming phenotypes. The cellular fatty acid profile was also observed to be affected by RssA and RssB. SFA-dependent inhibition of swarming was also observed in Proteus mirabilis, suggesting that either SFAs per se or the modulation of cellular fatty acid composition and hence homeostasis of membrane fluidity may be a conserved mechanism for regulating swarming motility in gram-negative bacteria.

Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin.

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive β-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.

A Mobile Quorum-Sensing System in Serratia marcescens

Quorum-sensing systems that have been widely identified in bacteria play important roles in the regulation of bacterial multicellular behavior by which bacteria sense population density to control various biological functions, including virulence. One characteristic of the luxIR quorum-sensing genes is their diverse and discontinuous distribution among proteobacteria. Here we report that the spnIR quorum-sensing system identified in the enterobacterium Serratia marcescens strain SS-1 is carried in a transposon, TnTIR, which has common characteristics of Tn3 family transposons and is mobile between chromosomes and plasmids of different enterobacterial hosts. SpnIR functions in the new host and was shown to negatively regulate the TnTIR transposition frequency. This finding may help reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way that we perceive regulation of bacterial multicellular behavior.

Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase

Using a component of the Escherichia coli protein degradation machinery, we have established a system to regulate protein stability in mycobacteria. A protein tag derived from the E. coli SsrA degradation signal did not affect several reporter proteins in wild-type Mycobacterium smegmatis or Mycobacterium tuberculosis. Expression of the adaptor protein SspB, which recognizes this modified tag and helps deliver tagged proteins to the protease ClpXP, strongly decreased the activities and protein levels of different reporters. This inactivation did not occur when the function of ClpX was inhibited. Using this system, we constructed a conditional M. smegmatis knockdown mutant in which addition of anhydrotetracycline (atc) caused depletion of the beta subunit of RNA polymerase, RpoB. The impact of atc on this mutant was dose-dependent. Very low amounts of atc did not prevent growth but increased sensitivity to an antibiotic that inactivates RpoB. Intermediate amounts of RpoB knockdown resulted in bacteriostasis and a more substantial depletion led to a decrease in viability by up to 99%. These studies identify SspB-mediated proteolysis as an efficient approach to conditionally inactivate essential proteins in mycobacteria. They further demonstrate that depletion of RpoB by ∼93% is sufficient to cause death of M. smegmatis.

Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating Pyruvate Dehydrogenase Activity

The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin(Sm)) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin(Sm) gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin(Sm) gene in S. marcescens CH-1. The S. marcescens CH-1 pirin(Sm) gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin(Sm) mutant. Concomitantly, the cellular NADH/NAD(+) ratio increased in the pirin(Sm) mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin(Sm) gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin(Sm) is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.

Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis

Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.

Characterization of the dapA-nlpB Genetic Locus Involved in Regulation of Swarming Motility, Cell Envelope Architecture, Hemolysin Production, and Cell Attachment Ability in Serratia marcescens

ABSTRACT Swarming migration of Serratia marcescens requires both flagellar motility and cellular differentiation and is a population-density-dependent behavior. While the flhDC and quorum-sensing systems have been characterized as important factors regulating S. marcescens swarming, the underlying molecular mechanisms are currently far from being understood. Serratia swarming is thermoregulated and is characterized by continuous surface migration on rich swarming agar surfaces at 30°C but not at 37°C. To further elucidate the mechanisms, identification of specific and conserved regulators that govern the initiation of swarming is essential. We performed transposon mutagenesis to screen for S. marcescens strain CH-1 mutants that swarmed at 37°C. Analysis of a “precocious-swarming” mutant revealed that the defect in a conserved dapASm-nlpBSm genetic locus which is closely related to the synthesis of bacterial cell wall peptidoglycan is responsible for the aberrant swarming phenotype. Further complementation and gene knockout studies showed that nlpBSm, which encodes a membrane lipoprotein, NlpBSm, but not dapASm, is specifically involved in swarming regulation. On the other hand, dapASm but not nlpBSm is responsible for the determination of cell envelope architecture, regulation of hemolysin production, and cellular attachment capability. While the nlpBSm mutant showed similar cytotoxicity to its parent strain, the dapASm mutant significantly increased in cytotoxicity. We present evidence that DapASm is involved in the determination of cell-envelope-associated phenotypes and that NlpBSm is involved in the regulation of swarming motility.