Use of an oxygen planar optode to assess the effect of high velocity microsprays on oxygen penetration in a human dental biofilms in-vitro.

BACKGROUND: Dental plaque biofilms are the causative agents of caries, gingivitis and periodontitis. Both mechanical and chemical strategies are used in routine oral hygiene strategies to reduce plaque build-up. If allowed to mature biofilms can create anoxic microenvironments leading to communities which harbor pathogenic Gram-negative anaerobes. When subjected to high velocity fluid jets and sprays biofilms can be fluidized which disrupts the biofilm structure and allows the more efficient delivery of antimicrobial agents. METHODS: To investigate how such jets may disrupt anoxic niches in the biofilm, we used planar optodes to measure the dissolved oxygen (DO) concentration at the base of in-vitro biofilms grown from human saliva and dental plaque. These biofilms were subject to "shooting" treatments with a commercial high velocity microspray (HVM) device. RESULTS: HVM treatment resulted in removal of much of the biofilm and a concurrent rapid shift from anoxic to oxic conditions at the base of the surrounding biofilm. We also assessed the impact of HVM treatment on the microbial community by tracking 7 target species by qPCR. There was a general reduction in copy numbers of the universal 16S RNA by approximately 95%, and changes of individual species in the target region ranged from approximately 1 to 4 log reductions. CONCLUSION: We concluded that high velocity microsprays removed a sufficient amount of biofilm to disrupt the anoxic region at the biofilm-surface interface.

Correction to: Use of an oxygen planar optode to assess the effect of high velocity microsprays on oxygen penetration in a human dental biofilms in-vitro.

An amendment to this paper has been published and can be accessed via the original article.

Streptococcus mutans yidC1 and yidC2 Impact Cell Envelope Biogenesis, the Biofilm Matrix, and Biofilm Biophysical Properties.

Proper envelope biogenesis of Streptococcus mutans, a biofilm-forming and dental caries-causing oral pathogen, requires two paralogs (yidC1 and yidC2) of the universally conserved YidC/Oxa1/Alb3 family of membrane integral chaperones and insertases. The deletion of either paralog attenuates virulence in vivo, but the mechanisms of disruption remain unclear. Here, we determined whether the deletion of yidC affects cell surface properties, extracellular glucan production, and/or the structural organization of the exopolysaccharide (EPS) matrix and biophysical properties of S. mutans biofilm. Compared to the wild type, the ΔyidC2 mutant lacked staining with fluorescent vancomycin at the division septum, while the ΔyidC1 mutant resembled the wild type. Additionally, the deletion of either yidC1 or yidC2 resulted in less insoluble glucan synthesis but produced more soluble glucans, especially at early and mid-exponential-growth phases. Alteration of glucan synthesis by both mutants yielded biofilms with less dry weight and insoluble EPS. In particular, the deletion of yidC2 resulted in a significant reduction in biofilm biomass and pronounced defects in the spatial organization of the EPS matrix, thus modifying the three-dimensional (3D) biofilm architecture. The defective biofilm harbored smaller bacterial clusters with high cell density and less surrounding EPS than those of the wild type, which was stiffer in compression yet more susceptible to removal by shear. Together, our results indicate that the elimination of either yidC paralog results in changes to the cell envelope and glucan production that ultimately disrupts biofilm development and EPS matrix structure/composition, thereby altering the physical properties of the biofilms and facilitating their removal. YidC proteins, therefore, represent potential therapeutic targets for cariogenic biofilm control.IMPORTANCE YidC proteins are membrane-localized chaperone insertases that are universally conserved in all bacteria and are traditionally studied in the context of membrane protein insertion and assembly. Both YidC paralogs of the cariogenic pathogen Streptococcus mutans are required for proper envelope biogenesis and full virulence, indicating that these proteins may also contribute to optimal biofilm formation in streptococci. Here, we show that the deletion of either yidC results in changes to the structure and physical properties of the EPS matrix produced by S. mutans, ultimately impairing optimal biofilm development, diminishing its mechanical stability, and facilitating its removal. Importantly, the universal conservation of bacterial yidC orthologs, combined with our findings, provide a rationale for YidC as a possible drug target for antibiofilm therapies.

Post-transcriptional regulation by distal Shine-Dalgarno sequences in the grpE-dnaK intergenic region of Streptococcus mutans.

A unique 373 bp region (igr66) between grpE and dnaK of Streptococcus mutans lacks a promoter but is required for optimal production of DnaK. Northern blotting using probes specific to hrcA, igr66 or dnaK revealed multiple transcripts produced from the dnaK operon and 5'-RACE mapped 5' termini of multiple dnaK transcripts within igr66. One product mapped to a predicted 5'-SL (stem-loop) and two others mapped just 5' to Shine-Dalgarno (SD)-like sequences located immediately upstream to dnaK and to a predicted SL 120 bp upstream of the dnaK start codon (3'-SL). A collection of cat reporter-gene strains containing mutant derivatives of igr66 were engineered. Chloramphenicol acetyltransferase (CAT) activity varied greatly between strains, but there were no correlative changes in cat mRNA levels. Interestingly, mutations introduced into the SD-like sequences 5' to the 3'-SL resulted in an 83-98% decrease in CAT activity. Markerless point mutations introduced upstream of dnaK in the SD-like sequences impaired growth at elevated temperatures and resulted in up to a 40% decrease in DnaK protein after heat shock. Collectively, these results indicate processing within igr66 enhances translation in a temperature dependent manner via non-canonical ribosome binding sites positioned >120 bp upstream of dnaK.

A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance.

Streptococcus mutans displays complex regulation of genetic competence, with ComX controlling late competence gene transcription. The rcrRPQ operon has been shown to link oxidative stress tolerance, (p)ppGpp metabolism and competence in S. mutans. Importantly, an rcrR polar (ΔrcrR-P) mutant is hyper-transformable, but an rcrR non-polar (ΔrcrR-NP) mutant cannot be transformed. Transcriptome comparisons of the rcrR mutants using RNA-Seq and quantitative real-time polymerase chain reaction revealed little expression in the 5' region of comX in ΔrcrR-NP, but high level expression in the 3' region. Northern blotting with comX probes revealed two distinct transcripts in the ΔrcrR-P and ΔrcrR-NP strains, and 5' Rapid Amplification of cDNA Ends mapped the 5' terminus of the shorter transcript to nt +140 of the comX structural gene, where a unique 69-aa open reading frame, termed XrpA, was encoded in a different reading frame than ComX. Two single-nucleotide substitution mutants (comX::T162C; comX::T210A) were introduced to disrupt XrpA without affecting the sequence of ComX. When the mutations were in the ΔrcrR-NP genetic background, ComX production and transformation were restored. Overexpression of xrpA led to impaired growth in aerobic conditions and decreased transformability. These results reveal an unprecedented mechanism for competence regulation and stress tolerance by a gene product encoded within the comX gene that appears unique to S. mutans.

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans.

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H2O2 via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)-ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.

YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, biofilm formation and cariogenicity of Streptococcus mutans.

The cariogenic bacterium Streptococcus mutans has two paralogues of the YidC/Oxa1/Alb3 family of membrane protein insertases/chaperones. Disruption of yidC2 results in loss of genetic competence, decreased membrane-associated ATPase activity and stress sensitivity (acid, osmotic and oxidative). Elimination of yidC1 has less severe effects, with little observable effect on growth or stress sensitivity. To examine the respective roles of YidC1 and YidC2, a conditional expression system was developed allowing simultaneous elimination of both endogenous YidCs. The function of the YidC C-terminal tails was also investigated and a chimeric YidC1 protein appended with the C terminus of YidC2 enabled YidC1 to complement a ΔyidC2 mutant for stress tolerance, ATP hydrolysis activity and extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Elimination of yidC1 or yidC2 affected levels of extracellular proteins, including GtfB, GtfC and adhesin P1 (AgI/II, PAc), which were increased without YidC1 but decreased in the absence of YidC2. Both yidC1 and yidC2 were shown to contribute to S. mutans biofilm formation and to cariogenicity in a rat model. Collectively, these results provide evidence that YidC1 and YidC2 contribute to cell surface biogenesis and protein secretion in S. mutans and that differences in stress sensitivity between the ΔyidC1 and ΔyidC2 mutants stem from a functional difference in the C-termini of these two proteins.

Independent gene duplications of the YidC/Oxa/Alb3 family enabled a specialized cotranslational function.

YidC/Oxa/Alb3 family proteins catalyze the insertion of integral membrane proteins in bacteria, mitochondria, and chloroplasts, respectively. Unlike gram-negative organisms, gram-positive bacteria express 2 paralogs of this family, YidC1/SpoIIIJ and YidC2/YgjG. In Streptococcus mutans, deletion of yidC2 results in a stress-sensitive phenotype similar to that of mutants lacking the signal recognition particle (SRP) protein translocation pathway, while deletion of yidC1 has a less severe phenotype. In contrast to eukaryotes and gram-negative bacteria, SRP-deficient mutants are viable in S. mutans; however, double SRP-yidC2 mutants are severely compromised. Thus, YidC2 may enable loss of the SRP by playing an independent but overlapping role in cotranslational protein insertion into the membrane. This is reminiscent of the situation in mitochondria that lack an SRP pathway and where Oxa1 facilitates cotranslational membrane protein insertion by binding directly to translation-active ribosomes. Here, we show that OXA1 complements a lack of yidC2 in S. mutans. YidC2 also functions reciprocally in oxa1-deficient Saccharomyces cerevisiae mutants and mediates the cotranslational insertion of mitochondrial translation products into the inner membrane. YidC2, like Oxa1, contains a positively charged C-terminal extension and associates with translating ribosomes. Our results are consistent with a gene-duplication event in gram-positive bacteria that enabled the specialization of a YidC isoform that mediates cotranslational activity independent of an SRP pathway.

Membrane proteomic analysis reveals overlapping and independent functions of Streptococcus mutans Ffh, YidC1, and YidC2.

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild-type strain and four mutants devoid of protein translocation machinery components, specifically ∆ffh, ∆yidC1, ∆yidC2, or ∆ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane-localized chaperone insertases. Our results suggest that the co-translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane-localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ∆yidC2 background may be non-functional, that ∆yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ∆ffh and ∆yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ∆yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1.

Functional overlap but lack of complete cross-complementation of Streptococcus mutans and Escherichia coli YidC orthologs.

Oxa/YidC/Alb family proteins are chaperones involved in membrane protein insertion and assembly. Streptococcus mutans has two YidC paralogs. Elimination of yidC2, but not yidC1, results in stress sensitivity with decreased membrane-associated F(1)F(o) ATPase activity and an inability to initiate growth at low pH or high salt concentrations (A. Hasona, P. J. Crowley, C. M. Levesque, R. W. Mair, D. G. Cvitkovitch, A. S. Bleiweis, and L. J. Brady, Proc. Natl. Acad. Sci. USA 102:17466-17471, 2005). We now show that Escherichia coli YidC complements for acid tolerance, and partially for salt tolerance, in S. mutans lacking yidC2 and that S. mutans YidC1 or YidC2 complements growth in liquid medium, restores the proton motive force, and functions to assemble the F(1)F(o) ATPase in a previously engineered E. coli YidC depletion strain (J. C. Samuelson, M. Chen, F. Jiang, I. Moller, M. Wiedmann, A. Kuhn, G. J. Phillips, and R. E. Dalbey, Nature 406:637-641, 2000). Both YidC1 and YidC2 also promote membrane insertion of known YidC substrates in E. coli; however, complete membrane integrity is not fully replicated, as evidenced by induction of phage shock protein A. While both function to rescue E. coli growth in broth, a different result is observed on agar plates: growth of the YidC depletion strain is largely restored by 247YidC2, a hybrid S. mutans YidC2 fused to the YidC targeting region, but not by a similar chimera, 247YidC1, nor by YidC1 or YidC2. Simultaneous expression of YidC1 and YidC2 improves complementation on plates. This study demonstrates functional redundancy between YidC orthologs in gram-negative and gram-positive organisms but also highlights differences in their activity depending on growth conditions and species background, suggesting that the complete functional spectrum of each is optimized for the specific bacteria and environment in which they reside.

Differential oxidative stress tolerance of Streptococcus mutans isolates affects competition in an ecological mixed-species biofilm model.

Streptococcus mutans strongly influences the development of pathogenic biofilms associated with dental caries. Our understanding of S. mutans behaviour in biofilms is based on a few well-characterized laboratory strains; however, individual isolates vary widely in genome content and virulence-associated phenotypes, such as biofilm formation and environmental stress sensitivity. Using an ecological biofilm model, we assessed the impact of co-cultivation of several S. mutans isolates with Streptococcus oralis and Actinomyces naeslundii on biofilm composition following exposure to sucrose. The laboratory reference strain S. mutans UA159 and clinical isolates Smu44 (most aciduric), Smu56 (altered biofilm formation) and Smu81 (more sensitive to oxidative stress) were used. Our data revealed S. mutans isolates varied in their ability to compete and become dominant in the biofilm after the addition of sucrose, and this difference correlated with sensitivity to H2 O2 produced by S. oralis. Smu81 was particularly sensitive to H2 O2 and could not compete with S. oralis in mixed-species biofilm, despite forming robust biofilms on its own. Thus, diminished oxidative stress tolerance in S. mutans isolates can impair their ability to compete in complex biofilms, even in the presence of sucrose, which could influence the progression of a healthy biofilm community to one capable of causing disease.

Phylogenomics and the dynamic genome evolution of the genus Streptococcus.

The genus Streptococcus comprises important pathogens that have a severe impact on human health and are responsible for substantial economic losses to agriculture. Here, we utilize 46 Streptococcus genome sequences (44 species), including eight species sequenced here, to provide the first genomic level insight into the evolutionary history and genetic basis underlying the functional diversity of all major groups of this genus. Gene gain/loss analysis revealed a dynamic pattern of genome evolution characterized by an initial period of gene gain followed by a period of loss, as the major groups within the genus diversified. This was followed by a period of genome expansion associated with the origins of the present extant species. The pattern is concordant with an emerging view that genomes evolve through a dynamic process of expansion and streamlining. A large proportion of the pan-genome has experienced lateral gene transfer (LGT) with causative factors, such as relatedness and shared environment, operating over different evolutionary scales. Multiple gene ontology terms were significantly enriched for each group, and mapping terms onto the phylogeny showed that those corresponding to genes born on branches leading to the major groups represented approximately one-fifth of those enriched. Furthermore, despite the extensive LGT, several biochemical characteristics have been retained since group formation, suggesting genomic cohesiveness through time, and that these characteristics may be fundamental to each group. For example, proteolysis: mitis group; urea metabolism: salivarius group; carbohydrate metabolism: pyogenic group; and transcription regulation: bovis group.

Phenotypic heterogeneity of genomically-diverse isolates of Streptococcus mutans.

High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.