Sphingolipids (SLs) play essential roles in most eukaryotes, but in the trypanosomatid protozoan Leishmania major their functions differ significantly. Previously we showed that null mutants defective in de novo sphingoid base synthesis (spt2−) lacked SLs but grew well and retained lipid rafts while replicating as promastigotes in vitro. However, they experienced catastrophic defects in membrane trafficking on entry into stationary phase, and failed to differentiate to the infective metacyclic form. Here we showed this mutant retained the ability to enter macrophages silently and inhibit activation, although as expected most parasites were destroyed. However, in mouse infections, after a delay rapidly progressive lesions appeared, and purified amastigotes were fully virulent to macrophages and mice. Mass spectrometry of spt2− amastigote lipids revealed the presence of high levels of parasite-specific inositol phosphorylceramides (IPCs) not synthesized by the mammalian hosts. Inhibitor studies showed that salvage occurs at the level of complex SLs, suggesting that parasites carry out ‘headgroup’ remodelling. Additionally, we describe a new defect of the spt2− promastigotes involving ‘empty’ acidocalcisomes (ACs), which may point to the origin of this organelle from the lysosome-related organelle/multivesicular body biogenesis pathway. However, ACs in spt2− amastigotes appeared quantitatively and morphologically normal. Thus salvage of SLs and other molecules by intracellular amastigotes play key roles in AC biogenesis and parasite survival in the host.
Enzymatic heme catabolism by heme oxygenases is conserved from bacteria to humans and proceeds through a common mechanism leading to the formation of iron, carbon monoxide, and biliverdin. The first members of a novel class of heme oxygenases were recently identified in Staphylococcus aureus (IsdG and IsdI) and were termed the IsdG-family of heme oxygenases. Enzymes of the IsdG-family form tertiary structures distinct from those of the canonical heme oxygenase family, suggesting that IsdG-family members degrade heme via a unique reaction mechanism. Herein we report that the IsdG-family of heme oxygenases degrade heme to the oxo-bilirubin chromophore staphylobilin. We also present the crystal structure of heme-bound IsdI in which heme ruffling and constrained binding of oxygen is consistent with cleavage of the porphyrin ring at the β– or γ–meso carbons. Combined, these data establish that the IsdG-family of heme oxygenases degrade heme to a novel chromophore distinct from biliverdin.
Staphylococcus aureus; heme oxygenase
In bloodstream-form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [3H]-inositol led us to hypothesize that de novo synthesis was responsible for supplying myo-inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate-limiting step of the de novo synthesis is the isomerization of glucose 6-phosphate to 1-d-myo-inositol-3-phosphate, catalysed by a 1-d-myo-inositol-3-phosphate synthase (INO1). When grown under non-permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream-form T. brucei. It also showed that the de novo synthesized myo-inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo-inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non-permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo-inositol and not a general inositol-less death. These results suggest that there is a distinction between de novo synthesized myo-inositol and that from the extracellular environment.
The recent reports of artemisinin (ART) resistance in the Thai-Cambodian border area raise a serious concern on the long-term efficacy of ARTs. To elucidate the resistance mechanisms, we performed in vitro selection with dihydroartemisinin (DHA) and obtained two parasite clones from Dd2 with more than 25-fold decrease in susceptibility to DHA. The DHA-resistant clones were more tolerant of stressful growth conditions and more resistant to several commonly used antimalarial drugs than Dd2. The result is worrisome since many of the drugs are currently used as ART partners in malaria control. This study showed that the DHA resistance is not limited to ring stage, but also occurred in trophozoites and schizonts. Microarray and biochemical analyses revealed pfmdr1 amplification, elevation of the antioxidant defense network, and increased expression of many chaperones in the DHA-resistant parasites. Without drug pressure, the DHA resistant parasites reverted to sensitive in approximately eight weeks, accompanied by de-amplification of pfmdr1 and reduced antioxidant activities. The parallel decrease and increase in pfmdr1 copy number and antioxidant activity and the up and down of DHA sensitivity strongly suggest that pfmdr1 and antioxidant defense play a role in in vitro resistance to DHA, providing potential molecular markers for ART resistance.
artemisinin resistance; drug selection; mechanism; pfmdr1; antioxidant defense
Trypanosoma brucei is the causative agent of African sleeping sickness, putting at risk up to 50 million people in sub-Saharan Africa. Current drug therapies are limited by toxicity and difficult treatment regimes and as the development of vaccines remains unlikely, the identification of better drugs to control this deadly disease is needed. Strategies for the identification of new lead compounds include phenotypic screening or target-based approaches. Implementation of the latter has been hampered by the lack of defined targets that are both essential and druggable. In this issue of Molecular Microbiology, Jones et al. report on the characterization of T. brucei pyridoxal kinase (PdxK), an enzyme required for the salvage of vitamin B6, an essential enzymatic cofactor. Genetic knockdown and small molecule inhibitor studies were used to demonstrate that PdxK is essential for parasite growth both in vitro and in a mouse model, providing both genetic and chemical validation of the target. An enzyme assay compatible with high throughput screening (HTS) was developed and the X-ray crystal structure solved, showing the potential for species selective inhibition. These studies add a greatly needed additional target into the drug discovery pipeline for this deadly parasitic infection.
Bacterial pathogens use cell-surface-associated adhesion molecules to promote host attachment and colonization, and the ability to modulate adhesion expression is critical to pathogen success. Here, we show that the human-specific pathogen the group A Streptococcus (GAS) uses a small regulatory RNA (sRNA) to regulate the expression of adhesive pili. The fibronectin / fibrinogen-binding / haemolytic-activity / streptokinase-regulator-X (FasX) sRNA, previously shown to positively regulate expression of the secreted virulence factor streptokinase (SKA), negatively regulates the production of pili on the GAS cell surface. FasX base-pairs to the extreme 5’ end of mRNA from the pilus biosynthesis operon, and this RNA:RNA interaction reduces the stability of the mRNA, while also inhibiting translation of at least the first gene in the pilus biosynthesis operon (cpa, which encodes a minor pilin protein). The negative regulation of pilus expression by FasX reduces the ability of GAS to adhere to human keratinocytes. Our findings cement FasX sRNA as an important regulator of virulence factor production in GAS and identify that FasX uses at least three distinct mechanisms, positive (ska mRNA) and negative (pilus operon mRNA) regulation of mRNA stability, and negative regulation of mRNA translation (cpa mRNA), to post-transcriptionally regulate target mRNAs during infection.
sRNA; Streptococcus pyogenes; pili; regulation; virulence factor; adherence
In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulators is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a protein complex with the sensor kinase SaeS and activate the sensor kinase’s phosphatase activity. Efficient activation of the phosphatase activity required the presence of both SaeP and SaeQ. When SaeP and SaeQ were ectopically expressed, the expression of coagulase, a sae target with low affinity for phosphorylated SaeR, was greatly reduced, while the expression of alpha-hemolysin, a sae target with high affinity for phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by the SaeRS TCS, these results suggest that the SaeRS TCS returns to the pre-activation state by a negative feedback mechanism.
Bacteria; Membrane proteins; Lipoproteins; Negative-feedback; Phosphatase
The two pathogenic species within the genus Neisseria cause the diseases gonorrhea and meningitis. While vaccines are available to protect against four N. meningitidis serogroups, there is currently no commercial vaccine to protect against serogroup B or against N. gonorrhoeae. Moreover, the available vaccines have significant limitations and with antibiotic resistance becoming an alarming issue, the search for effective vaccine targets to elicit long-lasting protection against Neisseria species is becoming more urgent. One strategy for vaccine development has targeted the neisserial iron import systems. Without iron, the Neisseriae cannot survive and therefore these iron import systems tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both gonococcal and meningococcal infections. These efforts have been boosted by recent reports of the crystal structures of the neisserial receptor proteins TbpA and TbpB, each solved in complex with human transferrin, an iron binding protein normally responsible for delivering iron to human cells. Here, we review the recent structural reports and put them into perspective with available functional studies in order to derive the mechanism(s) for how the pathogenic Neisseriae are able to hijack human iron transport systems for their own survival and pathogenesis.
Streptococcus mutans regulates genetic competence through a complex network that receives inputs from a number of environmental stimuli, including two signaling peptides designated as CSP and XIP. The response of the downstream competence genes to these inputs shows evidence of stochasticity and bistability and has been difficult to interpret. We have used microfluidic, single-cell methods to study how combinations of extracellular signals shape the response of comX, an alternative sigma factor governing expression of the late competence genes. We find that the composition of the medium determines which extracellular signal (XIP or CSP) can elicit a response from comX and whether that response is unimodal or bimodal across a population of cells. In a chemically defined medium, exogenous CSP does not induce comX, whereas exogenous XIP elicits a comX response from all cells. In complex medium, exogenous XIP does not induce comX, whereas CSP elicits a bimodal comX response from the population. Interestingly, bimodal behavior required an intact copy of comS, which encodes the precursor of XIP. The comS-dependent capability for both unimodal and bimodal response suggests that a constituent – most likely peptides – of complex medium interacts with a positive feedback loop in the competence regulatory network.
single-cell; bistability; quorum sensing; gene regulation; feedback; transformation
Carbapenems such as meropenem are being investigated for their potential therapeutic utility against highly drug-resistant tuberculosis. These β-lactams target the transpeptidases that introduce interpeptide cross-links into bacterial peptidoglycan thereby controlling rigidity of the bacterial envelope. Treatment of M. tuberculosis (Mtb) with the β-lactamase inhibitor clavulanate together with meropenem resulted in rapid, polar, cell lysis releasing cytoplasmic contents. In Mtb it has been previously demonstrated that 3-3 cross-linkages (involving two diaminopimelate (DAP) molecules) predominate over 4-3 cross-linkages (involving one DAP and one D-alanine) in stationary-phase cells. We purified and analyzed peptidoglycan from Mtb and found that 3-3 cross-linkages predominate throughout all growth phases and the ratio of 4-3/3-3 linkages does not vary significantly under any growth condition. Meropenem treatment was accompanied by a dramatic accumulation of unlinked pentapeptide stems with no change in the tetrapeptide pools, suggesting that meropenem inhibits both a D,D-carboxypeptidase and an L,D-transpeptidase. We purified a candidate D,D-carboxypeptidase DacB2 and showed that meropenem indeed directly inhibits this enzyme by forming a stable adduct at the enzyme active site. These results suggest that the rapid lysis of meropenem-treated cells is the result of synergistically inhibiting the transpeptidases that introduce 3,3-cross-links while simultaneously limiting the pool of available substrates available for cross-linking.
In many streptococci, quorum sensing utilizes secreted, linear peptides that engage cognate receptors to coordinate gene expression among members of a local population. Streptococcus mutans employs the secreted peptides CSP and XIP to stimulate production of antimicrobial bacteriocins and to induce development of competence for genetic transformation. Recent progress in the field reveals that pathways not only monitor the presence of signal emitters but also sense environmental factors. Both kinds of information are integrated by regulatory networks that then generate multiple outcomes, even among parallel cells growing in identical conditions. In this issue of Molecular Microbiology, Son and coworkers investigate how two medium types shape cellular responses to CSP and XIP pheromones in individuals across a population (Son et al., 2012). Their findings characterize restrictive properties of media differing in peptidic fragment content and reveal unusual signaling properties that contribute to bimodal responses of gene expression.
Quorum Sensing; Pheromones; Bacteriocins; Mutacins; Protease; Bimodal distribution; CDM
Infections with the azole-refractory yeast Candida glabrata are now commonly treated with the echinocandins caspofungin (CSF) or micafungin (MCF). True resistance (>32-fold decreased susceptibility) to these lipopeptide inhibitors of cell wall synthesis is rare and strictly associated with mutations in integral membrane proteins Fks1 or Fks2. In contrast, mutants exhibiting 4 to 32-fold CSF reduced susceptibility (CRS) were readily selected in vitro, and surprisingly demonstrated 4 to 32-fold MCF increased susceptibility (MIS). Sequencing and gene deletion demonstrated that CRS-MIS is Fks-independent. To explore alternative mechanisms, we initially employed Saccharomyces cerevisiae, and observed that CRS was conferred by multiple mutations (fen1Δ, sur4Δ, cka2Δ, and tsc10-ts) disrupting sphingolipid biosynthesis. Following this lead, C. glabrata fen1Δ and cka2Δ deletants were constructed, and shown to exhibit CRS-MIS. Sphingolipid analysis of CRS-MIS laboratory mutants and clinical isolates demonstrated elevated dihydrosphingosine (DHS) and phytosphingosine (PHS) levels, and consistent with this sequencing revealed fen1, sur4, ifa38, and sur2 mutations. Moreover, exogenous DHS or PHS conferred a CRS-MIS phenotype on wild-type C. glabrata. Exogenous PHS failed, however, to suppress CRS-MIS in a sur2 mutant blocked in conversion of DHS to PHS, implying that accumulation of these intermediates confers CRS-MIS. We conclude that membrane sphingolipids modulate echinocandin-Fks interaction.
echinocandin; caspofungin; micafungin; Candida glabrata; sphingolipid; Fks1
Mycobacterium tuberculosis CRPMt, encoded by Rv3676 (crp), is a CRP-like transcription factor that binds with the serC – Rv0885 intergenic region. In the present study, we evaluated CRPMt’s regulation of serC and Rv0885 in M. tuberculosis and M. bovis BCG, using site-specific mutagenesis, promoter fusions and RT-PCR. The CRPMt binding site was required for full expression of serC and Rv0885, and expression of both genes was reduced in M. tuberculosis and M. bovis BCG crp mutants. These data show that CRPMt binding directly activates both serC and Rv0885 expression. M. tuberculosis serC restored the ability of an Escherichia coli serC mutant to grow in serine-dropout medium, demonstrating that M. tuberculosis serC encodes a phosphoserine aminotransferase. Serine supplementation, or overexpression of serC, accelerated the growth of M. tuberculosis and M. bovis BCG crp mutants in mycomedium, but not within macrophages. These results establish a role for CRPMt in the regulation of amino acid biosynthesis, and show that reduced serine production contributes to the slow-growth phenotype of M. tuberculosis and M. bovis BCG crp mutants in vitro. Restoration of serine biosynthesis by serC expression will facilitate identification of additional CRPMt-regulated factors required by M. tuberculosis during macrophage and host infection.
Transformation requires specialized proteins to facilitate the binding and uptake of DNA. The genes of the B. subtilis comG operon (comGA–G) are required for transformation and to assemble a structure, the pseudopilus, in the cell envelope. No role for the pseudopilus has been established and the functions of the individual comG genes are unknown. We show that among the comG genes, only comGA is absolutely required for DNA binding to the cell surface. ComEA, an integral membrane DNA binding protein plays a minor role in the initial binding step, while an unidentified protein which communicates with ComGA must be directly responsible for binding to the cell. We show that the use of resistance to DNAse to measure “DNA uptake” reflects the movement of transforming DNA to a protected state in which it is not irreversibly associated with the protoplast, and presumably resides outside the cell membrane, in the periplasm or associated with the cell wall. We suggest that ComGA is needed for the acquisition of DNAse-resistance as well as for the binding of DNA to the cell surface. Finally, we show that the pseudopilus is required for DNA uptake and we offer a revised model for the transformation process.
Transformation; Bacillus subtilis; secretion ATPase; DNA uptake; ComGA
Transformable (competent) cells of Bacillus subtilis are blocked in cell division because the traffic ATPase ComGA prevents the formation of FtsZ rings. Although ComGA-deficient cells elongate and form FtsZ rings, cell division remains blocked at a later stage and the cells become mildly filamented. Here we show that the highly conserved protein Maf is synthesized predominantly in competent cells under the direct control of the transcription factor ComK and is solely responsible for the later block in cell division. In vivo and in vitro data show that Maf binds to both ComGA and DivIVA. A point mutation in maf that interferes with Maf binding to DivIVA also interferes with the ability of Maf to inhibit cell division. Based on these findings, we propose that Maf and ComGA mediate mechanisms for the inhibition of cell division in competent cells with Maf acting downstream of ComGA. We further suggest that Maf must interact with DivIVA to inhibit cell division.
Transformation; Cell division; Bacillus subtilis; ComGA; Maf
In Candida albicans, a fungal pathogen, the small G-protein Ras1 regulates many important behaviors including white-opaque switching, biofilm formation, and the induction and maintenance of hyphal growth. Like other Ras proteins, Ras1 is activated upon guanine triphosphate binding, and its activity is further modulated by post-translational lipid modifications. Here, we report that the levels of membrane-associated, full-length Ras1 were higher in hyphae than in yeast, and that yeast contained a shorter, soluble Ras1 species that resulted from cleavage. Deletion of the putative cleavage site led to more rapid induction of hyphal growth and delayed hypha-to-yeast transitions. The cleaved Ras1 species was less able to activate its effector, adenylate cyclase (Cyr1), unless tethered to the membrane by a heterologous membrane-targeting domain. Ras1 cleavage was repressed by cAMP-signaling, indicating the presence of a positive feedback loop in which Cyr1 and cAMP influence Ras1. The C. albicans quorum sensing molecule farnesol, which inhibits Cyr1 and represses filamentation, caused an increase in the fraction of Ras1 in the cleaved form, particularly in nascent yeast formed from hyphae. This newly recognized mode of Ras regulation may control C. albicans Ras1 activity in important ways.
Candida albicans; Ras1; Cyr1; farnesol; morphology
Candida albicans hypha formation which has been stimulated via the Ras1-cAMP-Efg1 signalling cascade is inhibited by farnesol, a C. albicans autoregulatory factor, and small molecules such as dodecanol. In cultures containing farnesol or dodecanol, hypha formation was restored upon addition of dibutyryl-cAMP. The CAI4-Ras1G13V strain, which carries a dominant-active variant of Ras1 and forms hyphae in the absence of inducing stimuli, grew as yeast in medium with farnesol or dodecanol; the heat shock sensitivity of the CAI4-Ras1G13V strain was also suppressed by these compounds. Neither Pde1 nor Pde2 was necessary for the repression of hyphal growth by farnesol or dodecanol. Two transcripts, CTA1 and HSP12, which are at higher levels upon mutation of Ras1 or Cdc35, were increased in abundance in cells grown with farnesol or dodecanol. Microscopic analysis of strains carrying CTA1 and HWP1 promoter fusions grown with intermediate concentrations of farnesol or dodecanol indicated a link between cells with the increased expression of cAMP-repressed genes and cells repressed for hypha formation. Because several cAMP-controlled outputs are affected by farnesol and dodecanol, our findings suggest that these compounds impact activity of the Ras1-Cdc35 pathway, thus leading to an alteration of C. albicans morphology.
Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome-mediated respiration. This reduction was explained by RNAi-mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function.
The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod-shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA-EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.
The production and removal of regulatory RNAs must be controlled to ensure proper physiological responses. SsrA RNA (tmRNA), a regulatory RNA conserved in all bacteria, is cell-cycle regulated and is important for control of cell cycle progression in Caulobacter crescentus. We report that RNase R, a highly conserved 3’–5’ exoribonuclease, is required for the selective degradation of SsrA RNA in stalked cells. Purified RNase R degrades SsrA RNA in vitro, and is kinetically competent to account for all SsrA RNA turnover. SmpB, a tmRNA-binding protein, protects SsrA RNA from RNase R degradation in vitro, and the levels of SmpB protein during the cell cycle correlate with SsrA RNA stability. These results suggest that SmpB binding controls the timing of SsrA RNA degradation by RNase R. We propose a model for the regulated degradation of SsrA RNA in which RNase R degrades SsrA RNA from a non-tRNA-like 3’ end, and SmpB specifically protects SsrA RNA from RNase R. This model explains the regulation of SsrA RNA in other bacteria, and suggests that a highly conserved regulatory mechanism controls SsrA activity.
Caulobacter; cell cycle; ribonuclease; RNA-binding protein; tmRNA
A perplexing aspect of fungal secondary metabolite gene clusters is that most clusters remain ‘silent’ under common laboratory growth conditions where activation is obtained through gene manipulation or encounters with environmental signals. Few proteins have been found involved in repression of silent clusters. Through multicopy suppressor mutagenesis, we have identified a novel cluster suppressor in Aspergillus nidulans, MvlA (modulator of veA
loss). Genetic assessment of MvlA mutants revealed the role of both itself and VeA (but not the VeA partner LaeA) in the suppression of the cryptic ors gene cluster producing orsellinic acid and its F9775 derivatives. Loss of veA upregulates F9775A and F9775B production and this increase is reduced 4~5 fold when an overexpression mvlA (OE::mvlA) allele is introduced into the ΔveA background. Previous studies have implicated a positive role for GcnE (H3K9 acetyltransferase of the SAGA/ADA complex) in ors cluster expression and here we find expression of gcnE is upregulated in ΔveA and suppressed by OE::mvlA in the ΔveA background. H3K9 acetylation levels of ors cluster genes correlated with gcnE expression and F9775 production in ΔveA and OE::mvlAΔveA strains. Finally, deletion of gcnE in the ΔveA background abolishes ors cluster activation and F9775 production. Together, this work supports a role for VeA and MvlA in modifying SAGA/ADA complex activity.
LaeA; Velvet Complex; F9775A; Urc4; suppressor; CclA; SAGA/ADA
Bacteria isolated from marine sponges, including the Silicibacter-Ruegeria (SR) subgroup of the Roseobacter clade, produce N-acylhomoserine lactone (AHL) quorum sensing signal molecules. This study is the first detailed analysis of AHL quorum sensing in sponge-associated bacteria, specifically Ruegeria sp. KLH11, from the sponge Mycale laxissima. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated ssaRI, ssbRI, and sscI (sponge-associated symbiont locus A, B, and C, luxRI or luxI homologue). SsaI produced predominantly long-chain 3-oxo-AHLs and both SsbI and SscI specified 3-OH-AHLs. Addition of exogenous AHLs to KLH11 increased the expression of ssaI but not ssaR, ssbI or ssbR, and genetic analyses revealed a complex interconnected arrangement between SsaRI and SsbRI systems. Interestingly, flagellar motility was abolished in the ssaI and ssaR mutants, with the flagellar biosynthesis genes under strict SsaRI control, and active motility only at high culture density. Conversely, ssaI and ssaR mutants formed more robust biofilms than wild type KLH11. AHLs and transcript of the ssaI gene were detected in M. laxissima extracts suggesting that AHL signaling contributes to the decision between motility and sessility and that it also may facilitate acclimation to different environments including the sponge host.
quorum sensing; acyl-homoserine lactones; LuxI-LuxR type regulation; sponge symbionts; motility; biofilm
The DNA phage ΦX174 encodes the integral membrane protein E whose expression leads to host cell lysis by inhibition of the peptidoglycan synthesis enzyme MraY. Here we use mutagenesis to characterize the molecular details of the E lysis mechanism. We find that a minimal 18-residue region with the modified wild-type sequences of the conserved transmembrane helix of E is sufficient to lyse host cells and that specific residues within and at the boundaries of this helix are important for activity. This suggests that positioning of the helix in the membrane is critical for interactions with MraY. We further characterize the interaction site of the transmembrane helix with MraY demonstrating E forms a stable complex with MraY. Triggering cell lysis by peptidoglycan synthesis inhibition is a traditional route for antimicrobial strategies. Understanding the mechanism of bacterial cell lysis by E will provide insights into new antimicrobial strategies using re-engineered E peptides.
MraY; alanine scanning; antibiotics; cell wall; peptidoglycan; microvirus
Escherichia coli uses the DsbA/DsbB system for introducing disulfide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulfide bond formation but does not entirely eliminate it. Whether such background disulfide bond forming activity is enzyme-catalyzed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulfide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulfide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulfide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulfenic acid form and reacts with substrate proteins to form mixed disulfide adducts. DsbC either prevents the formation of these mixed disulfides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyze disulfide bond formation. Although this PspE/DsbC system is not responsible for the background disulfide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.
rhodanese; disulfide bond formation; protein disulfide isomerase; sulfenic acid; DsbA; DsbC
The binding site of the cyclic peptide antibiotics capreomycin and viomycin is located on the ribosomal subunit interface close to nucleotides C1409 in 16S rRNA and C1920 in 23S rRNA. In Mycobacterium tuberculosis, the 2′-hydroxyls of both nucleotides are methylated by the enzyme TlyA. Loss of these methylations through inactivation of TlyA confers resistance to capreomycin and viomycin. We report here that TlyA orthologs occur in diverse bacteria and fall into two distinct groups. One group, now termed TlyAI, has shorter N- and C-termini and methylates only C1920; the second group (now TlyAII) includes the mycobacterial enzyme, and these longer orthologs methylate at both C1409 and C1920. Ribosomal subunits are the preferred substrates for both groups of orthologs. Amino acid substitutions at the N-terminus of TlyAII reduce its ability to methylate these substrates. Growing pairs of recombinant TlyAII
Escherichia coli strains in competition shows that even subtle changes in the level of rRNA methylation lead to significant differences in susceptibility to sub-inhibitory concentrations of capreomycin. The findings reveal that 2′-O-methyls at both C1409 and C1920 play a role in facilitating the inhibitory effects of capreomycin and viomycin on the bacterial ribosome.
rRNA; 2′-O-methylation; rRNA methyltransferase; mRNA translation; ribosomal subunit interface; capreomycin/viomycin