Biology subjects

Burne, R. A.

Publications and source records attributed to Burne, R. A..

6 recordsLinked to original sources

bioRxiv2018

Species Designations Belie Phenotypic and Genotypic Heterogeneity in Oral Streptococci

Health-associated oral Streptococcus species are promising probiotic candidates to protect against dental caries. Ammonia production through the arginine deiminase system (ADS), which can increase the pH of oral biofilms, and direct antagonism of caries-associated bacterial species are desirable properties for oral probiotic strains. ADS and antagonistic activities can vary dramatically among individuals, but the genetic basis for these differences is unknown. We sequenced whole genomes of a diverse set of clinical oral Streptococcus isolates and examined the genetic basis of variability in ADS and antagonistic activities. A total of 113 isolates were included and represented ten species: S. australis, A12-like, S. cristatus, S. gordonii, S. intermedius, S. mitis, S. oralis including S. oralis subsp. dentisani, S. parasanguinis, S. salivarius, and S. sanguinis. Mean ADS activity and antagonism on Streptococcus mutans UA159 were measured for each isolate, and each isolate was whole genome-shotgun sequenced on an Illumina MiSeq. Phylogenies were built of genes known to be involved in ADS activity and antagonism. Several approaches to correlate the pan-genome with phenotypes were performed. Phylogenies of genes previously identified in ADS activity and antagonism grouped isolates by species but not by phenotype. GWAS identified additional genes potentially involved in ADS activity or antagonism across all the isolates we sequenced as well as within several species. Phenotypic heterogeneity in oral streptococci is not necessarily reflected by genotype and is not species-specific. Probiotic strains must be carefully selected based on characterization of each strain, and not based on inclusion within a certain species.\n\nImportanceRepresentative type strains are commonly used to characterize bacterial species, yet species are phenotypically and genotypically heterogeneous. Conclusions about strain physiology and activity based on a single strain therefore may be inappropriate and misleading. When selecting strains for probiotic use, the assumption that all strains within a species share the same desired probiotic characteristics share those characteristics may result in selection of a strain that lacks the desired traits, and therefore makes a minimally effective or ineffective probiotic. Health-associated oral streptococci are promising candidates for anti-caries probiotics, but strains need to be carefully selected based on observed phenotypes. We characterized the genotype and anti-caries phenotypes of strains from ten species of oral Streptococci and demonstrate poor correlation between genotype and phenotype across all species.

microbiology↗

bioRxiv2018

Essential Roles of the sppRA Fructose-Phosphate Phosphohydrolase Operon in Carbohydrate Metabolism and Virulence Expression by Streptococcus mutans

The dental caries pathogen Streptococcus mutans can ferment a variety of sugars to produce organic acids. Exposure of S. mutans to certain non-metabolizable carbohydrates such as xylitol impairs growth and can cause cell death. Recently, the presence of a sugar-phosphate stress in S. mutans was demonstrated using a mutant lacking 1-phosphofructokinase (FruK) that accumulates fructose-1-phosphate (F-1-P). Here we studied an operon in S. mutans, sppRA, which was highly expressed in the fruK mutant. Biochemical characterization of a recombinant SppA protein indicated that it possessed hexose-phosphate phosphohydrolase activity, with preferences for F-1-P and, to a lesser degree, fructose-6-phosphate (F-6-P). SppA activity was stimulated by Mg2+ and Mn2+, but inhibited by NaF. SppR, a DeoR-family regulator, repressed the expression of the sppRA operon to minimum levels in the absence of the fructose-derived metabolites, F-1-P and likely also F-6-P. Accumulation of F-1-P, as a result of growth on fructose, not only induced sppA expression, it significantly altered biofilm maturation through increased cell lysis and enhanced extracellular DNA release. Constitutive expression of sppA, via a plasmid or by deleting sppR, greatly alleviated fructose-induced stress in a fruK mutant, enhanced resistance to xylitol, and reversed effects of fructose on biofilm formation. Finally, by identifying three additional putative phosphatases that are capable of promoting sugar-phosphate tolerance, we show that S. mutans is capable of mounting a sugar-phosphate stress response by modulating the levels of certain glycolytic intermediates, functions that are interconnected with the ability of the organism to manifest key virulence behaviors.\n\nImportanceStreptococcus mutans is a major etiologic agent for dental caries, primarily due to its ability to form biofilms on tooth surface and to convert carbohydrates into organic acids. We have discovered a two-gene operon in S. mutans that regulates fructose metabolism by controlling the levels of fructose-1-phosphate, a potential signaling compound that affects bacterial behaviors. With fructose becoming increasingly common and abundant in the human diet, we reveal the ways fructose may alter bacterial development, stress tolerance, and microbial ecology in the oral cavity to promote oral diseases.

microbiology↗

bioRxiv2018

Preferred hexoses influence long-term memory and induction of lactose catabolism by Streptococcus mutans

Bacteria prioritize sugar metabolism via carbohydrate catabolite repression, which regulates global gene expression to optimize the catabolism of preferred substrates. Here, we report an unusual long-term memory effect in certain Streptococcus mutans strains that alters adaptation to growth on lactose after prior exposure to glucose or fructose. In strain GS-5, cells that were first cultured on fructose then transferred to lactose displayed an exceptionally long lag (>11 h) and slower growth, compared to cells first cultured on glucose or cellobiose, which displayed a reduction in lag phase by as much as 10 h. Mutants lacking the cellobiose-PTS or phospho-{beta}-glucosidase lost the accelerated growth on lactose associated with prior culturing on glucose. The memory effects of glucose or fructose on lactose catabolism were not as profound in strain UA159, but the lag phase was considerably shorter in mutants lacking the glucose-PTS EIIMan. Interestingly, when S. mutans was cultivated on lactose, significant quantities of free glucose accumulated in the medium, with higher levels found in the cultures of strains lacking EIIMan, glucokinase, or both. Free glucose was also detected in cultures that were utilizing cellobiose or trehalose, albeit at lower levels. Such release of hexoses by S. mutans is likely of biological significance as it was found that cells required small amounts of glucose or other preferred carbohydrates to initiate efficient growth on lactose. These findings suggest that S. mutans modulates the induction of lactose utilization based on its prior exposure to glucose or fructose, which can be liberated from common disaccharides.\n\nIMPORTANCEUnderstanding the molecular mechanisms employed by oral bacteria to control sugar metabolism is key to developing novel therapies for management of dental caries and other oral diseases. Lactose is a naturally occurring disaccharide that is abundant in dairy products and commonly ingested by humans. However, for the dental caries pathogen Streptococcus mutans, relatively little is known about the molecular mechanisms that regulate expression of genes required for lactose uptake and catabolism. Two peculiarities of lactose utilization by S. mutans are explored here: a) S. mutans excretes glucose that it cleaves from lactose and b) prior exposure to certain carbohydrates can result in a long-term inability to use lactose. The study begins to shed light on how S. mutans may bet-hedge to optimize its persistence and virulence in the human oral cavity.

microbiology↗

bioRxiv2018

Intracellular signaling through the comRS system in Streptococcus mutans genetic competence

AbstractEntry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS signaling system, which consists of the cytosolic receptor ComR and the 7-residue signal peptide XIP, which is derived from ComS. Extracellular XIP imported by an oligopeptide permease interacts with ComR to form a transcriptional activator for both comX and comS. Therefore, extracellular XIP can function as an exogenous signal to trigger S. mutans competence. However, the mechanisms that process ComS and export it as XIP are not fully known in S. mutans. The observation that comX is expressed bimodally under some environmental conditions suggests that ComR may also interact with endogenously produced XIP or ComS, creating an intracellular positive feedback loop in comS transcription. Here we use single cell and microfluidic methods to compare the effects of the native comS gene and extracellular XIP on comX expression. We find that deletion of comS reduces the response of comX to extracellular XIP. We also find that comS-overexpressing cells autoactivate their comX even when their growth medium is rapidly exchanged, although this autoactivation requires an intact copy of comS under control of its own promoter. However comS-overexpressing cells do not activate comS-deficient mutants growing in coculture. These data show that individual cells can activate comX without exporting or importing the XIP or ComS signal, and that endogenously and exogenously produced ComS/XIP have inequivalent effects on comX behavior. These data are fully consistent with a model in which intracellular positive feedback in comS transcription plays a role in ComRS signaling, and is responsible for the bimodal expression of comX.\n\nAuthor SummaryHeterogeneous gene expression in genetically identical populations plays an important role in bacterial persistence and survival under changing environmental conditions. In the oral pathogen Streptococcus mutans, the physiological state of genetic competence can exhibit bimodality, with only some cells becoming competent. S. mutans controls its entry into competence by using the ComRS signaling system to activate comX, a gene encoding the master competence regulator ComX. The ComRS system is understood as a quorum sensing system, in which the extracellular accumulation of the small signal peptide XIP, derived from ComS, induces comX expression. We coupled observation of bacteria that fluoresce when comX is active with mathematical analysis and chemical binding assays to show that activation of comX does not necessarily require extracellular XIP or ComS, and that comX-active cells do not necessarily export XIP. Our experiments and mathematical modeling indicate that a positive feedback loop in comS transcription allows a cell to activate comX in response to its own XIP or ComS in the absence of extracellular XIP, or to amplify its comX response to extracellular XIP if present. Such positive feedback loops are often the cause of bimodal gene expression like that seen in S. mutans competence.

microbiology↗

bioRxiv2018

Threshold regulation and stochasticity from the MecA/ClpCP proteolytic system in Streptococcus mutans competence

Many bacterial species use the MecA/ClpCP proteolytic system to block entry into genetic competence. In Streptococcus mutans, MecA/ClpCP degrades ComX (also called SigX), an alternative sigma factor for the comY operon and other late competence genes. Although the mechanism of MecA/ClpCP has been studied in multiple Streptococcus species, its role within noisy competence pathways is poorly understood. S. mutans competence can be triggered by two different peptides, CSP and XIP, but it is not known whether MecA/ClpCP acts similarly for both stimuli, how it affects competence heterogeneity, and how its regulation is overcome. We have studied the effect of MecA/ClpCP on the activation of comY in individual S. mutans cells. Our data show that MecA/ClpCP is active under both XIP and CSP stimulation, that it provides threshold control of comY, and that it adds noise in comY expression. Our data agree quantitatively with a model in which MecA/ClpCP prevents adventitious entry into competence by sequestering or intercepting low levels of ComX. Competence is permitted when ComX levels exceed a threshold, but cell-to-cell heterogeneity in MecA levels creates variability in that threshold. Therefore MecA/ClpCP provides a stochastic switch, located downstream of the already noisy comX, that enhances phenotypic diversity.

microbiology↗

bioRxiv2017

Intercellular communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses and ecological competitiveness. One example is activation of natural genetic competence by ComR and the comX-inducing peptide (XIP) in Streptococcus mutans. Although the competence pathway can be activated by addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signal molecule has not been rigorously tested. Co-culture model systems were developed that included a \"sender\" strain that overexpressed the XIP precursor (ComS) and a \"responder\" strain harboring a GFP reporter fusion to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using i) planktonic culture systems, ii) cells suspended in an agarose matrix or iii) cells growing in biofilms. XIP was shown to be freely diffusible and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with endogenously-produced peptide(s) in populations in various environments and physiologic states.\n\nIMPORTANCEThe comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids and addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence and evolution of a ubiquitous human pathogen and related organisms.

microbiology↗