Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans.

The cariogenic pathogen Streptococcus mutans contains two CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA transcripts (pre-crRNA) containing repeats and spacers to mature crRNA guides. In this study, we determined the crystal structure of SmuCas5c at a resolution of 1.72 Å, which revealed the presence of an N-terminal modified RNA recognition motif and a C-terminal twisted ß-sheet domain with four bound sulphate molecules. Analysis of surface charge and residue conservation of the SmuCas5c structure suggested the location of an RNA-binding site in a shallow groove formed by the RNA recognition motif domain with several conserved positively charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134). Purified SmuCas5c exhibited metal-independent ribonuclease activity against single-stranded pre-CRISPR RNAs containing a stem-loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the repeat sequence at a single cleavage site located at the 3'-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based mutational analysis revealed that the conserved residues Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic residues. In addition, site-directed mutagenesis of positively charged residues Lys52, Arg109, and Arg134 located near the catalytic triad had strong negative effects on the RNase activity of this protein, suggesting that these residues are involved in RNA binding. Taken together, our results reveal functional diversity of Cas5c ribonucleases and provide further insight into the molecular mechanisms of substrate selectivity and activity of these enzymes.

K+ modulates genetic competence and the stress regulon of Streptococcus mutans.

Potassium (K+) is the most abundant cation in dental plaque fluid. Previously, we reported the link between K+ transport via Trk2 in Streptococcus mutans and its two critical virulence attributes: acid tolerance and surface adhesion. Herein, we build further on the intimate link between K+ levels and S. mutans biology. High (>25 mM) versus low (≤5 mM) K+ concentrations in the growth medium affected conformational epitopes of cell surface-localized adhesin P1. At low K+, the expression of stress response elements gcrR and codY, cell-adhesion-associated genes such as spaP and metabolism-associated genes such as bglP was induced at stationary phase (P<0.05), suggesting that K+-mediated regulation is growth phase-dependent and stress-sensitive. Production of the newly discovered secretory protein encoded by SMU_63c was strongly dependent on the availability of K+ and growth phase. This protein is a newly discovered regulator of genetic competence and biofilm cell density. Thus, the influence of K+ on DNA transformation efficiency was also examined. Compared with 25 mM K+ concentration, the presence of low K+ reduced the transformation frequency by 100-fold. Genetic transformation was abolished in a strain lacking a Trk2 system under all K+ concentrations tested. Consistent with these findings, repression of competence-associated genes, comS and comX, was observed under low environmental K+ conditions and in the strain lacking Trk2. Taken together, these results highlight a pivotal role for environmental K+ as a regulatory cation that modulates stress responses and genetic transformation in S. mutans.

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c.

Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1- and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance.

UNLABELLED: Potassium (K(+)) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K(+) and a variety of K(+) transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K(+) acquisition in Streptococcus mutans and the importance of K(+) homeostasis for its virulence attributes. The S. mutans genome harbors four putative K(+) transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K(+) cotransporter (GlnQHMP), and a channel-like K(+) transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K(+)] less than 5 mM eliminated biofilm formation in S. mutans. The functionality of the Trk2 system was confirmed by complementing an Escherichia coli TK2420 mutant strain, which resulted in significant K(+) accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K(+)-dependent cellular response of S. mutans to environment stresses. IMPORTANCE: Biofilm formation and stress tolerance are important virulence properties of caries-causing Streptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment of S. mutans. K(+) is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K(+) transporters in S. mutans. We identified the most important system for K(+) homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K(+) for the activity of biofilm-forming enzymes, which explains why such high levels of K(+) would favor biofilm formation.

Mechanistic insights revealed by the crystal structure of a histidine kinase with signal transducer and sensor domains.

Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.

The copYAZ Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans.

UNLABELLED: In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the copYABZ operon for copper homeostasis, where copA and copB encode copper-transporting P-type ATPases, whereas copY and copZ regulate the expression of the cop operon. Streptococcus mutans is a biofilm-forming oral pathogen that harbors a putative copper-transporting copYAZ operon. Here, we characterized the role of copYAZ operon in the physiology of S. mutans and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in S. mutans cells by generating oxidative stress and disrupting their membrane potential. Deletion of the copYAZ operon in S. mutans strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of S. mutans to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the gtfB, gtfC, gtfD, gbpB, and gbpC genes, whose products have roles in maintaining the structural and/or functional integrity of the S. mutans biofilm. Furthermore, supplementation with copper or loss of copYAZ resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the ΔcopYAZ strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of S. mutans. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the copYAZ-null mutant. Taking these results collectively, we have established the function of the S. mutans CopYAZ system in copper export and have further expanded knowledge on the importance of copper homeostasis and the CopYAZ system in modulating streptococcal physiology, including stress tolerance, membrane potential, genetic competence, and biofilm formation. IMPORTANCE: S. mutans is best known for its role in the initiation and progression of human dental caries, one of the most common chronic diseases worldwide. S. mutans is also implicated in bacterial endocarditis, a life-threatening inflammation of the heart valve. The core virulence factors of S. mutans include its ability to produce and sustain acidic conditions and to form a polysaccharide-encased biofilm that provides protection against environmental insults. Here, we demonstrate that the addition of copper and/or deletion of copYAZ (the copper homeostasis system) have serious implications in modulating biofilm formation, stress tolerance, and genetic transformation in S. mutans. Manipulating the pathways affected by copper and the copYAZ system may help to develop potential therapeutics to prevent S. mutans infection in and beyond the oral cavity.

Molecular and structural basis of glutathione import in Gram-positive bacteria via GshT and the cystine ABC importer TcyBC of Streptococcus mutans.

Glutathione (GSH) protects cells against oxidative injury and maintains a range of vital functions across all branches of life. Despite recent advances in our understanding of the transport mechanisms responsible for maintaining the spatiotemporal homeostasis of GSH and its conjugates in eukaryotes and Gram-negative bacteria, the molecular and structural basis of GSH import into Gram-positive bacteria has remained largely uncharacterized. Here, we employ genetic, biochemical and structural studies to investigate a possible glutathione import axis in Streptococcus mutans, an organism that has hitherto served as a model system. We show that GshT, a type 3 solute binding protein, displays physiologically relevant affinity for GSH and glutathione disulfide (GSSG). The crystal structure of GshT in complex with GSSG reveals a collapsed structure whereby the GS-I-leg of GSSG is accommodated tightly via extensive interactions contributed by the N- and C-terminal lobes of GshT, while the GS-II leg extends to the solvent. This can explain the ligand promiscuity of GshT in terms of binding glutathione analogues with substitutions at the cysteine-sulfur or the glycine-carboxylate. Finally, we show that GshT primes glutathione import via the L-cystine ABC transporter TcyBC, a membrane permease, which had previously exclusively been associated with the transport of L-cystine.

[Functional analysis of VicK kinase activity in Streptococcus mutans].

OBJECTIVE: To investigate the regulatory function on physiology and virulence of VicK kinase activity in Streptococcus mutans. METHODS: PCR ligation mutagenesis was used to construct a vicK knock-out mutant, and kinase activity abolished VicK was expressed by a streptococcal vector in this vicK null mutant. Colony morphology, overnight culture, biofilm formation and gene expression involved in biofilm formation were analyzed. Delta VicK, strains harboring a complemented wild-type vicK, and a vector without insert were used as controls. RESULTS: Colonies of VicK(H217A) were smoother and more elevated than that of wild-type UA159 and complementary strain SMCVicK; cells from VicK(H217A) overnight culture coaggregated on the bottom of glass tubes; no obvious alteration was observed in VicK(H217A) biofilm; expressions of gbpB, ftf, gtfD were repressed while gtfB/C were up-regulated (P < 0.05). CONCLUSION: VicK kinase activity is important for maintaining normal growth, biofilm formation and expression of genes involved in biofilm formation in Streptococcus mutans.

Oligomerization of the response regulator ComE from Streptococcus mutans is affected by phosphorylation.

We have previously characterized the interactions of the response regulator ComE from Streptococcus mutans and DNA binding sites through DNase I footprinting and electrophoretic mobility shift assay analysis. Since response regulator functions are often affected by their phosphorylation state, we investigated how phosphorylation affects the biochemical function of ComE. Unlike many response regulators, we found that the phosphorylation state of ComE does not likely play a role in DNA binding affinity but rather seems to induce the formation of an oligomeric form of the protein. The role of this oligomerization state for ComE function is discussed.

Streptococcus mutans Proteases Degrade Dentinal Collagen.

Here, we explored the role of S. mutans's whole cell and discrete fractions in the degradation of type I collagen and dentinal collagen. Type I collagen gels and human demineralized dentin slabs (DS) were incubated in media alone or with one of the following: overnight (O/N) or newly inoculated (NEW) cultures of S. mutans UA159; intracellular proteins, supernatant or bacterial membranes of O/N cultures. Media from all groups were analyzed for protease-mediated release of the collagen-specific imino acid hydroxyproline. Images of type I collagen and DS were analyzed, respectively. Type I collagen degradation was highest for the supernatant (p < 0.05) fractions, followed by intracellular components and O/N cultures. Collagen degradation for DS samples was highest for O/N samples, followed by supernatant, and intracellular components (p < 0.05). There was lower detectable degradation for both type I collagen and DS from NEW culture samples (p < 0.05), and there was no type I collagen or DS degradation detected for bacterial membrane samples. Structural changes to type I collagen gel and dentinal collagen were observed, respectively, following incubation with S. mutans cultures (O/N and NEW), intracellular components, and supernatant. This study demonstrates that intracellular and extracellular proteolytic activities from S. mutans enable this cariogenic bacterium to degrade type I and dentinal collagen in a growth-phase dependent manner, potentially contributing to the progression of dental caries.

Regulation of the competence pathway as a novel role associated with a streptococcal bacteriocin.

The oral biofilm organism Streptococcus mutans must face numerous environmental stresses to survive in its natural habitat. Under specific stresses, S. mutans expresses the competence-stimulating peptide (CSP) pheromone known to induce autolysis and facilitate the uptake and incorporation of exogenous DNA, a process called DNA transformation. We have previously demonstrated that the CSP-induced CipB bacteriocin (mutacin V) is a major factor involved in both cellular processes. Our objective in this work was to characterize the role of CipB bacteriocin during DNA transformation. Although other bacteriocin mutants were impaired in their ability to acquire DNA under CSP-induced conditions, the ΔcipB mutant was the only mutant showing a sharp decrease in transformation efficiency. The autolysis function of CipB bacteriocin does not participate in the DNA transformation process, as factors released via lysis of a subpopulation of cells did not contribute to the development of genetic competence in the surviving population. Moreover, CipB does not seem to participate in membrane depolarization to assist passage of DNA. Microarray-based expression profiling showed that under CSP-induced conditions, CipB regulated ∼130 genes, among which are the comDE locus and comR and comX genes, encoding critical factors that influence competency development in S. mutans. We also discovered that the CipI protein conferring immunity to CipB-induced autolysis also prevented the transcriptional regulatory activity of CipB. Our data suggest that besides its role in cell lysis, the S. mutans CipB bacteriocin also functions as a peptide regulator for the transcriptional control of the competence regulon.

Characterization of DNA binding sites of the ComE response regulator from Streptococcus mutans.

In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

Characterization of a glutamate transporter operon, glnQHMP, in Streptococcus mutans and its role in acid tolerance.

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans.

Peptide alarmone signalling triggers an auto-active bacteriocin necessary for genetic competence.

The induction of genetic competence is a strategy used by bacteria to increase their genetic repertoire under stressful environmental conditions. Recently, Streptococcus pneumoniae has been shown to co-ordinate the uptake of transforming DNA with fratricide via increased expression of the peptide pheromone responsible for competence induction. Here, we document that environmental stress-induced expression of the peptide pheromone competence-stimulating peptide (CSP) in the oral pathogen Streptococcus mutans. We showed that CSP is involved in the stress response and determined the CSP-induced regulon in S. mutans by microarray analysis. Contrary to pneumococcus, S. mutans responds to increased concentrations of CSP by cell lysis in only a fraction of the population. We have focused on the mechanism of cell lysis and have identified a novel bacteriocin as the 'death effector'. Most importantly, we showed that this bacteriocin causes cell death via a novel mechanism of action: intracellular action against self. We have also identified the cognate bacteriocin immunity protein, which resides in a separate unlinked genetic locus to allow its differential regulation. The role of the lytic response in S. mutans competence is also discussed. Together, these findings reveal a novel autolytic pathway in S. mutans which may be involved in the dissemination of fitness-enhancing genes in the oral biofilm.

The LiaFSR system regulates the cell envelope stress response in Streptococcus mutans.

Maintaining cell envelope integrity is critical for bacterial survival, including bacteria living in a complex and dynamic environment such as the human oral cavity. Streptococcus mutans, a major etiological agent of dental caries, uses two-component signal transduction systems (TCSTSs) to monitor and respond to various environmental stimuli. Previous studies have shown that the LiaSR TCSTS in S. mutans regulates virulence traits such as acid tolerance and biofilm formation. Although not examined in streptococci, homologs of LiaSR are widely disseminated in Firmicutes and function as part of the cell envelope stress response network. We describe here liaSR and its upstream liaF gene in the cell envelope stress tolerance of S. mutans strain UA159. Transcriptional analysis established liaSR as part of the pentacistronic liaFSR-ppiB-pnpB operon. A survey of cell envelope antimicrobials revealed that mutants deficient in one or all of the liaFSR genes were susceptible to Lipid II cycle interfering antibiotics and to chemicals that perturbed the cell membrane integrity. These compounds induced liaR transcription in a concentration-dependent manner. Notably, under bacitracin stress conditions, the LiaFSR signaling system was shown to induce transcription of several genes involved in membrane protein synthesis, peptidoglycan biosynthesis, envelope chaperone/proteases, and transcriptional regulators. In the absence of an inducer such as bacitracin, LiaF repressed LiaR-regulated expression, whereas supplementing cultures with bacitracin resulted in derepression of liaSR. While LiaF appears to be an integral component of the LiaSR signaling cascade, taken collectively, we report a novel role for LiaFSR in sensing cell envelope stress and preserving envelope integrity in S. mutans.

Inactivation of VicK affects acid production and acid survival of Streptococcus mutans.

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.

Atypical roles for Campylobacter jejuni amino acid ATP binding cassette transporter components PaqP and PaqQ in bacterial stress tolerance and pathogen-host cell dynamics.

Campylobacter jejuni is a human pathogen causing severe diarrheal disease; however, our understanding of the survival of C. jejuni during disease and transmission remains limited. Amino acid ATP binding cassette (AA-ABC) transporters in C. jejuni have been proposed as important pathogenesis factors. We have investigated a novel AA-ABC transporter system, encoded by cj0467 to cj0469, by generating targeted deletions of cj0467 (the membrane transport component) and cj0469 (the ATPase component) in C. jejuni 81-176. The analyses described here have led us to designate these genes paqP and paqQ, respectively (pathogenesis-associated glutamine [q] ABC transporter permease [P] and ATPase [Q]). We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Altered resistance to a series of environmental and in vivo stresses was also observed: both mutants were hyperresistant to aerobic and organic peroxide stress, and while the DeltapaqP mutant was also hyperresistant to heat and osmotic shock, the DeltapaqQ mutant was more susceptible than the wild type to the latter two stresses. The DeltapaqP and DeltapaqQ mutants also displayed a surprising but statistically significant increase in recovery from macrophages and epithelial cells in short-term intracellular survival assays. Annexin V, 4',6-diamidino-2-phenylindole (DAPI), and Western blot analyses revealed that macrophages infected with the DeltapaqP or DeltapaqQ mutant exhibited transient but significant decreases in cell death and extracellular signal-regulated kinase-mitogen-activated protein kinase activation compared to levels in wild-type-infected cells. The DeltapaqP mutant was not defective in either short-term or longer-term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation of an AA-ABC transporter with bacterial stress tolerances and host cell responses to pathogen infection.

Role of Dilution Rate and Nutrient Availability in the Formation of Microbial Biofilms.

We revisited the mathematical model of the chemostat and examined consequences of considerably decreasing the concentration of limiting nutrient in the inflow for the growth of both the planktonic and biofilm cells in the chemostat tank (fermenter). The model predicts a substantially lower steady-state biomass of planktonic cells in response to decreasing inflowing nutrient concentration. Contrarily, the steady-state concentration of nutrient inside the fermenter is expected to remain the same, as long as the inflowing concentration does not fall below its value. This allows the biofilm cells to grow at a rate regulated only by the exchange rate of the medium (dilution rate). We maintained a strain of Enterococcus faecalis in a chemostat of our own design with limiting nutrient in the inflow set near saturation constant at three dilution rates (0.09, 0.28, and 0.81 h-1). The highest dilution rate was near the critical rate calculated by the model. The one-day total biofilm buildup was 21× larger and its estimated growth rate 2.4× higher at highest dilution rate than at the lowest one. This increased biofilm formation with increased dilution rates is in agreement with previously published data on pure and mixed continuous flow cultures.

Modification of Pseudomonas aeruginosa Pa5196 type IV Pilins at multiple sites with D-Araf by a novel GT-C family Arabinosyltransferase, TfpW.

Pseudomonas aeruginosa Pa5196 produces type IV pilins modified with unusual alpha1,5-linked d-arabinofuranose (alpha1,5-D-Araf) glycans, identical to those in the lipoarabinomannan and arabinogalactan cell wall polymers from Mycobacterium spp. In this work, we identify a second strain of P. aeruginosa, PA7, capable of expressing arabinosylated pilins and use a combination of site-directed mutagenesis, electrospray ionization mass spectrometry (MS), and electron transfer dissociation MS to identify the exact sites and extent of pilin modification in strain Pa5196. Unlike previously characterized type IV pilins that are glycosylated at a single position, those from strain Pa5196 were modified at multiple sites, with modifications of alphabeta-loop residues Thr64 and Thr66 being important for normal pilus assembly. Trisaccharides of alpha1,5-D-Araf were the principal modifications at Thr64 and Thr66, with additional mono- and disaccharides identified on Ser residues within the antiparallel beta sheet region of the pilin. TfpW was hypothesized to encode the pilin glycosyltransferase based on its genetic linkage to the pilin, weak similarity to membrane-bound GT-C family glycosyltransferases (which include the Mycobacterium arabinosyltransferases EmbA/B/C), and the presence of characteristic motifs. Loss of TfpW or mutation of key residues within the signature GT-C glycosyltransferase motif completely abrogated pilin glycosylation, confirming its involvement in this process. A Pa5196 pilA mutant complemented with other Pseudomonas pilins containing potential sites of modification expressed nonglycosylated pilins, showing that TfpW's pilin substrate specificity is restricted. TfpW is the prototype of a new type IV pilin posttranslational modification system and the first reported gram-negative member of the GT-C glycosyltransferase family.

Autoinducer-2-regulated genes in Streptococcus mutans UA159 and global metabolic effect of the luxS mutation.

Autoinducer 2 (AI-2) is the only species-nonspecific autoinducer known in bacteria and is produced by both gram-negative and gram-positive organisms. Consequently, it is proposed to function as a universal quorum-sensing signal for interaction between bacterial species. AI-2 is produced as the by-product of a metabolic transformation carried out by the LuxS enzyme. To separate the metabolic function of the LuxS enzyme from the signaling role of AI-2, we carried out a global transcriptome analysis of a luxS null mutant culture of Streptococcus mutans UA159, an important cariogenic bacterium and a crucial component of the dental plaque biofilm community, in comparison to a luxS null mutant culture supplemented with chemically pure 4,5-dihydroxy-2,3-pentanedione, the precursor of AI-2. The data revealed fundamental changes in gene expression affecting 585 genes (30% of the genome) which could not be restored by the signal molecule AI-2 and are therefore not caused by quorum sensing but by lack of the transformation carried out by the LuxS enzyme in the activated methyl cycle. All functional classes of enzymes were affected, including genes known to be important for biofilm formation, bacteriocin synthesis, competence, and acid tolerance. At the same time, 59 genes were identified whose transcription clearly responded to the addition of AI-2. Some of them were related to protein synthesis, stress, and cell division. Three membrane transport proteins were upregulated which are not related to any of the known AI-2 transporters. Three transcription factors were identified whose transcription was stimulated repeatedly by AI-2 addition during growth. Finally, a global regulatory protein, the delta subunit of the RNA polymerase (rpoE), was induced 147-fold by AI-2, representing the largest differential gene expression observed. The data show that many phenotypes related to the luxS mutation cannot be ascribed to quorum sensing and have identified for the first time regulatory proteins potentially mediating AI-2-based signaling in gram-positive bacteria.