Despite many examples of obligate epibiotic symbiosis (one organism living on the surface of another) in nature, such an interaction has rarely been observed between two bacteria. Here, we further characterize a newly reported interaction between a human oral obligate parasitic bacterium TM7x (cultivated member of Candidatus Saccharimonas formerly Candidate Phylum TM7), and its basibiont Actinomyces odontolyticus species (XH001), providing a model system to study epiparasitic symbiosis in the domain Bacteria. Detailed microscopic studies indicate that both partners display extensive morphological changes during symbiotic growth. XH001 cells manifested as short rods in monoculture, but displayed elongated and hyphal morphology when physically associated with TM7x. Interestingly, these dramatic morphological changes in XH001 were also induced in oxygen-depleted conditions, even in the absence of TM7x. Targeted qRT-PCR analyses revealed that both the physical association with TM7x as well as oxygen depletion triggered up-regulation of key stress response genes in XH001, and in combination, these conditions act in an additive manner. TM7x and XH001 co-exist with relatively uniform cell morphologies under nutrient-replete conditions. However, upon nutrient depletion, TM7x-associated XH001 displayed a variety of cell morphologies, including swollen cell body, clubbed ends and even cell lysis, and a large portion of TM7x cells transformed from ultrasmall cocci into elongated cells. Our study demonstrates a highly dynamic interaction between epibiont TM7x and its basibiont XH001 in response to physical association or environmental cues such as oxygen level and nutritional status, as reflected by their morphological and physiological changes during symbiotic growth.
Obligate; epibiont; symbiosis; bacterial interaction; TM7; Actinomyces
Denture stomatitis is a prevalent inflammatory condition of the mucosal tissue in denture wearers that is triggered by microorganisms. While Candida has been extensively studied for its role in stomatitis etiology, the bacterial component largely remains to be investigated. Our data show that certain types of bacteria are significantly associated with denture health and disease. Furthermore, the bacterial communities residing on the teeth and dentures of the same person are similar to each other independently of the surface, and thus, denture health could impact the maintenance of remaining teeth and vice versa.
While investigation of the microbiome on natural oral surfaces has generated a wealth of information, few studies have examined the microbial communities colonizing dentures and their relationship to oral health. To address this knowledge gap, we characterized the bacterial community associated with dentures and remaining teeth in healthy individuals and patients with denture stomatitis. The microbiome compositions of matched denture and tooth plaque samples of 10 healthy individuals and 9 stomatitis patients were determined by 16S rRNA gene pyrosequencing. The microbial communities colonizing dentures and remaining teeth in health and disease were very similar to each other. Matched denture and tooth samples from the same individuals shared a significantly higher percentage of identical phylotypes than random pairs of samples from different study participants. Despite these overall similarities, several bacterial phylotypes displayed discrete health- and stomatitis-associated denture colonization, while others were distinct in health and disease independently of the surface. Certain phylotypes exhibited differential colonization of dentures and teeth independently of denture health status. In conclusion, denture and natural tooth surfaces in health and stomatitis harbor similar bacterial communities. Individual-related rather than surface-specific factors play a significant role in the bacterial phylotype composition colonizing dentures and teeth. This individual-specific mutual influence on denture and tooth surface colonization could be an important factor in maintaining oral health in denture wearers. Discrete differences in colonization patterns for distinct genera and phylotypes warrant further studies regarding their potential involvement or utility as specific indicators of health and disease development in denture-wearing individuals.
IMPORTANCE Denture stomatitis is a prevalent inflammatory condition of the mucosal tissue in denture wearers that is triggered by microorganisms. While Candida has been extensively studied for its role in stomatitis etiology, the bacterial component largely remains to be investigated. Our data show that certain types of bacteria are significantly associated with denture health and disease. Furthermore, the bacterial communities residing on the teeth and dentures of the same person are similar to each other independently of the surface, and thus, denture health could impact the maintenance of remaining teeth and vice versa.
bacteria; denture; microbiome; stomatitis
We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.
Bacterial biofilm infections remain prevalent reasons for implant
failure. Dental implant placement occurs in the oral environment, which harbors
a plethora of biofilm-forming bacteria. Due to its trans-mucosal placement, part
of the implant structure is exposed to oral cavity and there is no effective
measure to prevent bacterial attachment to implant materials. Here, we
demonstrated that UV treatment of titanium immediately prior to use
(photofunctionalization) affects the ability of human polymicrobial oral biofilm
communities to colonize in the presence of salivary and blood components.
UV-treatment of machined titanium transformed the surface from hydrophobic to
superhydrophilic. UV-treated surfaces exhibited a significant reduction in
bacterial attachment as well as subsequent biofilm formation compared to
untreated ones, even though overall bacterial viability was not affected. The
function of reducing bacterial colonization was maintained on UV-treated
titanium that had been stored in a liquid environment before use. Denaturing
gradient gel-electrophoresis (DGGE) and DNA sequencing analyses revealed that
while bacterial community profiles appeared different between UV-treated and
untreated titanium in the initial attachment phase, this difference vanished as
biofilm formation progressed. Our findings confirm that
UV-photofunctionalization of titanium has a strong potential to improve outcome
of implant placement by creating and maintaining antimicrobial surfaces.
Photofunctionalization; titanium; bacteria; biofilm; peri-implantitis
Bacterial adherence is an essential virulence factor in pathogenesis and infection. Fusobacterium nucleatum has a central role in oral biofilm architecture by acting as a bridge between early Gram-positive and late Gram-negative colonizers that do not otherwise adhere to each other. In this study, we survey a key adherence interaction of F. nucleatum with Porphyromonas gingivalis, and present evidence that multiple fusobacterial adhesins have a role in the attachment of F. nucleatum ATCC 23726 to P. gingivalis in a highly strain-dependent manner. Interaction between these species displayed varying sensitivities to arginine, galactose and lactose. Arginine was found to hamper coaggregation by at least 62% and up to 89% with several P. gingivalis strains and galactose inhibition ranged from no inhibition up to 58% with the same P. gingivalis strains. Lactose consistently inhibited F. nucleatum interaction with these P. gingivalis strains ranging from 40% to 56% decrease in coaggregation. Among the adhesins involved are the previously described Fap2 and surprisingly, RadD, which was described in an earlier study for its function in attachment of F. nucleatum to Gram-positive species. We also provide evidence for the presence of at least one additional adhesin that is sensitive to arginine but unlike Fap2 and RadD, is not a member of the autotransporter family type of fusobacterial large outer membrane proteins. The strain-specific binding profile of multiple fusobacterial adhesins to P. gingivalis highlights the heterogeneity and complexity of interspecies interactions in the oral cavity.
adhesin; biofilm; coaggregation; Fusobacterium nucleatum; Porphyromonas gingivalis
Myxococcus xanthus performs coordinated social motility of cell groups through the extension and retraction of type IV pili (TFP) on solid surfaces, which requires both TFP and exopolysaccharides (EPS). By submerging cells in a liquid medium containing 1% methylcellulose, M. xanthus TFP-driven motility was induced in isolated cells and independently of EPS. We measured and analyzed the movements of cells using community tracking algorithms, which combine single-cell resolution with statistics from large sample populations. Cells without significant multi-cellular social interactions have surprisingly complex behaviors: EPS− cells exhibited a pronounced increase in the tendency to stand vertically and moved with qualitatively different characteristics than other cells. A decrease in the EPS secretion of cells correlates with a higher instantaneous velocity, but with lower directional persistence in trajectories. Moreover, EPS− cells do not adhere to the surface as strongly as wild-type and EPS overproducing cells, and display a greater tendency to have large deviations between the direction of movement and the cell axis, with cell velocity showing only minimal dependence on the direction of movement. The emerging picture is that EPS does not simply provide rheological resistance to a single mechanism but rather that the availability of EPS impacts motility pattern.
Streptococcus mutans is considered the principal cariogenic bacterium for dental caries. Despite the recognition of their importance for cariogenesis, the possible coordination among S. mutans’ main virulence factors, including glucan production, acidogenicity and aciduricity, has been less well studied. In the present study, using S. mutans strains with surface-displayed pH-sensitive pHluorin, we revealed sucrose availability- and Gtf functionality-dependent proton accumulation on S. mutans surface. Consistent with this, using a pH-sensitive dye, we demonstrated that both in vivo cell-produced and in vitro enzymatically synthesized insoluble glucans displayed proton-concentrating ability. Global transcriptomics revealed proton accumulation triggers the up-regulation of genes encoding functions involved in acid tolerance response in a glucan-dependent manner. Our data suggested that this proton enrichment around S. mutans could pre-condition the bacterium for acid-stress. Consistent with this hypothesis, we found S. mutans strains defective in glucan production were more acid sensitive. Our study revealed for the first time that insoluble glucans is likely an essential factor linking acidogenicity with aciduricity. The coordination of these key virulence factors could provide new insights on how S. mutans may have become a major cariogenic pathogen.
Dental caries, one of the most globally widespread infectious diseases, is intimately linked to pH dynamics. In supragingival plaque, after the addition of a carbohydrate source, bacterial metabolism decreases the pH which then subsequently recovers. Molecular mechanisms supporting this important homeostasis are poorly characterized in part due to the fact that there are hundreds of active species in dental plaque. Only a few mechanisms (for example, lactate fermentation, the arginine deiminase system) have been identified and studied in detail. Here, we conducted what is to our knowledge, the first full transcriptome and metabolome analysis of a diverse oral plaque community by using a functionally and taxonomically robust in vitro model system greater than 100 species. Differential gene expression analyses from the complete transcriptome of 14 key community members revealed highly varied regulation of both known and previously unassociated pH-neutralizing pathways as a response to the pH drop. Unique expression and metabolite signatures from 400 detected metabolites were found for each stage along the pH curve suggesting it may be possible to define healthy and diseased states of activity. Importantly, for the maintenance of healthy plaque pH, gene transcription activity of known and previously unrecognized pH-neutralizing pathways was associated with the genera Lactobacillus, Veillonella and Streptococcus during the pH recovery phase. Our in vitro study provides a baseline for defining healthy and disease-like states and highlights the power of moving beyond single and dual species applications to capture key players and their orchestrated metabolic activities within a complex human oral microbiome model.
The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect mRNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.
F. nucleatum; oral streptococci; interspecies interaction; RadD
It is well established that host-associated microbial communities can interfere with the colonization and establishment of microbes of foreign origins, a phenomenon often referred to as bacterial interference or colonization resistance. However, due to the complexity of the indigenous microbiota, it has been extremely difficult to elucidate the community colonization resistance mechanisms and identify the bacterial species involved. In a recent study, we have established an in vitro mice oral microbial community (O-mix) and demonstrated its colonization resistance against an Escherichia coli strain of mice gut origin. In this study, we further analyzed the community structure of the O-mix by using a dilution/regrowth approach and identified the bacterial species involved in colonization resistance against E. coli. Our results revealed that, within the O-mix there were three different types of bacterial species forming unique social structure. They act as ‘Sensor', ‘Mediator' and ‘Killer', respectively, and have coordinated roles in initiating the antagonistic action and preventing the integration of E. coli. The functional role of each identified bacterial species was further confirmed by E. coli-specific responsiveness of the synthetic communities composed of different combination of the identified players. The study reveals for the first time the sophisticated structural and functional organization of a colonization resistance pathway within a microbial community. Furthermore, our results emphasize the importance of ‘Facilitation' or positive interactions in the development of community-level functions, such as colonization resistance.
colonization resistance; microbial community; social structure; host-associated microbiota; facilitation
The human microbiome influences and reflects the health or disease state of the host. Periodontitis, a disease affecting about half of American adults, is associated with alterations in the subgingival microbiome of individual tooth sites. Although it can be treated, the disease can reoccur and may progress without symptoms. Without prognostic markers, follow-up examinations are required to assess reoccurrence and disease progression and to determine the need for additional treatments. To better identify and predict the disease progression, we aim to determine whether the subgingival microbiome can serve as a diagnosis and prognosis indicator. Using metagenomic shotgun sequencing, we characterized the dynamic changes in the subgingival microbiome in periodontitis patients before and after treatment at the same tooth sites. At the taxonomic composition level, the periodontitis-associated microorganisms were significantly shifted from highly correlated in the diseased state to poorly correlated after treatment, suggesting that coordinated interactions among the pathogenic microorganisms are essential to disease pathogenesis. At the functional level, we identified disease-associated pathways that were significantly altered in relative abundance in the two states. Furthermore, using the subgingival microbiome profile, we were able to classify the samples to their clinical states with an accuracy of 81.1%. Follow-up clinical examination of the sampled sites supported the predictive power of the microbiome profile on disease progression. Our study revealed the dynamic changes in the subgingival microbiome contributing to periodontitis and suggested potential clinical applications of monitoring the subgingival microbiome as an indicator in disease diagnosis and prognosis.
Periodontitis is a common oral disease. Although it can be treated, the disease may reoccur without obvious symptoms. Current clinical examination parameters are useful in disease diagnosis but cannot adequately predict the outcome of individual tooth sites after treatment. A link between the subgingival microbiota and periodontitis was identified previously; however, it remains to be investigated whether the microbiome can serve as a diagnostic and prognostic indicator. In this study, for the first time, we characterized the subgingival microbiome of individual tooth sites before and after treatment using a large-scale metagenomic analysis. Our longitudinal study revealed changes in the microbiota in taxonomic composition, cooccurrence of subgingival microorganisms, and functional composition. Using the microbiome profiles, we were able to classify the clinical states of subgingival plaque samples with a high accuracy. Follow-up clinical examination of sampled sites indicates that the subgingival microbiome profile shows promise for the development of diagnostic and prognostic tools.
Since dentures can serve as a reservoir for halitosis-causing oral bacteria, halitosis development is a concern for denture wearers. In this study, we surveyed the prevalence of four selected halitosis-related species (Fusobacterium nucleatum, Tannerella forsythia, Veillonella atypica and Klebsiella pneumoniae) in clinical denture plaque samples, and developed denture biofilm models for these species in vitro to facilitate assessment of antimicrobial treatment efficacy. Design : Denture plaque from ten healthy and ten denture stomatitis patients was screened for the presence of aforementioned four species by PCR. Biofilm formation by these halitosis-associated species on the surfaces of denture base resin (DBR) discs was evaluated by crystal violet staining and confocal laser scanning microscopy. The efficacy of denture cleanser treatment on these mono-species biofilms was evaluated by colony counting. Results : 80% of the subjects in the denture stomatitis group and 60% in the healthy group contained at least one of the targeted halitosis-related species in their denture plaque. All halitosis species tested were able to form biofilms on DBR disc surfaces to varying degrees. These in vitro mono-species resin biofilm models were used to evaluate the efficacy of denture cleansers, which exhibited differential efficacies. When forming biofilms on resin surfaces, the halitosis-related species displayed enhanced resistance to denture cleansers compared with their planktonic counterparts. Conclusion : The four selected halitosis-related bacterial species examined in this study are present on the majority of dentures. The mono-species biofilm models established on DBR discs for these species are an efficient screening tool for dental product evaluation.
Antimicrobial treatment; biofilms; denture; halitosis; model
Nutrient or niche-based competition among bacteria is a widespread phenomenon in natural environment. Such inter-species interactions are often mediated by secreted soluble factors and/or direct cell–cell contact. As ubiquitous soil bacteria, Myxococcus species are able to produce a variety of bioactive secondary metabolites to inhibit the growth of other competing bacterial species. Meanwhile, Myxococcus sp. also exhibit sophisticated predatory behavior, an extreme form of competition that is often stimulated by close contact with prey cells and largely depends on the availability of solid surfaces. Myxococcus sp. can also be isolated from aquatic environments. However, studies focusing on the interaction between Myxococcus and other bacteria in such environments are still limited. In this study, using the well-studied M. xanthus DK1622 and E. coli as model interspecies interaction pair, we demonstrated that in a aqueous environment, Myxococcus xanthus was able to kill Escherichia coli in a cell contact-dependent manner, and that the observed contact dependent killing required the formation of co-aggregates between M. xanthus and E. coli cells. Further analysis revealed that exopolysaccharide (EPS), type IV pilus (TFP) and lipopolysaccharide (LPS) mutants of M. xanthus displayed various degrees of attenuation in E. coli killing, and it correlated well with the mutants' reduction in EPS production. In addition, M. xanthus showed differential binding ability to different bacteria, and bacterial strains unable to co-aggregate with M. xanthus can escape the killing, suggesting the specific nature of co-aggregation and the targeted killing of interacting bacteria. In conclusion, our results demonstrated EPS mediated, contact-dependent killing of E. coli by M. xanthus, a strategy that might facilitate the survival of this ubiquitous bacterium in aquatic environments.
M. xanthus; contact-dependent interaction; co-aggregation; aqueous environment
Bacteria develop a broad range of phage resistance mechanisms, such as prevention of phage adsorption and CRISPR/Cas system, to survive phage predation. In this study, Pseudomonas aeruginosa PA1 strain was infected with lytic phage PaP1, and phage-resistant mutants were selected. A high percentage (~30%) of these mutants displayed red pigmentation phenotype (Red mutant). Through comparative genomic analysis, one Red mutant PA1r was found to have a 219.6 kb genomic fragment deletion, which contains two key genes hmgA and galU related to the observed phenotypes. Deletion of hmgA resulted in the accumulation of a red compound homogentisic acid; while A galU mutant is devoid of O-antigen, which is required for phage adsorption. Intriguingly, while the loss of galU conferred phage resistance, it significantly attenuated PA1r in a mouse infection experiment. Our study revealed a novel phage resistance mechanism via chromosomal DNA deletion in P. aeruginosa.
The classic organization by Socransky and coworkers categorized the oral bacteria of the subgingival plaque into different complexes. Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia are grouped into the red complex that is highly correlated with periodontal disease. Socransky's work closely associates red with orange complex species such as Fusobacterium nucleatum and Prevotella intermedia but not with members of the other complexes. While the relationship between species contained by these complexes is in part supported by their ability to physically attach to each other, the physiological consequences of these interactions and associations are less clear. In this study, we employed T. denticola as a model organism to analyze contact-dependent responses to interactions with species belonging to the same complex (P. gingivalis and T. forsythia), the closely associated orange complex (using F. nucleatum and P. intermedia as representatives) and the unconnected yellow complex (using Streptococcus sanguinis and S. gordonii as representatives). RNA was extracted from T. denticola alone as well as after pairwise co-incubation for 5 hrs with representatives of the different complexes, and the respective gene expression profiles were determined using microarrays. Numerous genes related to motility, metabolism, transport, outer membrane and hypothetical proteins were differentially regulated in T. denticola in the presence of the tested partner species. Further analysis revealed a significant overlap in the affected genes and we identified a general response to the presence of other species, those specific to two of the three complexes as well as individual complexes. Most interestingly, many predicted major antigens (e.g. flagella, Msp, CTLP) were suppressed in responses that included red complex species indicating that the presence of the most closely associated species induces immune-evasive strategies. In summary, the data presented here provide an in-depth understanding of the transcriptional responses triggered by contact-dependent interactions between microorganisms inhabiting the periodontal pocket.
Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram positive bacteria, including Bacillus and Lactococcus encode functions involved in protein post-export. In this study, we constructed a PrsA-deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/ membrane protein profile as well as cell surface related phenotypes, including auto-aggregation, increased surface hydrophobicity, and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface-display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to the ones identified in Bacillus, and thus is likely involved in protein post-export.
foldase protein PrsA; protein secretion; Streptococcus mutans
Exopolysaccharide (EPS) of Myxococcus xanthus is a well-regulated cell surface component. In addition to its known functions for social motility and fruiting body formation on solid surfaces, EPS has also been proposed to play a role in multi-cellular clumping in liquid medium, though this phenomenon has not been well studied. In this report, we confirmed that M. xanthus clumps formed in liquid were correlated with EPS levels and demonstrated that the EPS encased cell clumps exhibited biofilm-like structures. The clumps protected the cells at physiologically relevant EPS concentrations, while cells lacking EPS exhibited significant reduction in long-term viability and resistance to stressful conditions. However, excess EPS production was counterproductive to vegetative growth and viable cell recovery declined in extended late stationary phase as cells became trapped in the matrix of clumps. Therefore, optimal EPS production by M. xanthus is important for normal physiological functions in liquid.
Myxococcus xanthus; exopolysaccharide; vegetative growth; stress survival; stationary phase recovery
Our knowledge of microbial diversity in the human oral cavity has vastly expanded during the last two decades of research. However, much of what is known about the behavior of oral species to date derives from pure culture approaches and the studies combining several cultivated species, which likely does not fully reflect their function in complex microbial communities. It has been shown in studies with a limited number of cultivated species that early oral biofilm development occurs in a successional manner and that continuous low pH can lead to an enrichment of aciduric species. Observations that in vitro grown plaque biofilm microcosms can maintain similar pH profiles in response to carbohydrate addition as plaque in vivo suggests a complex microbial community can be established in the laboratory. In light of this, our primary goal was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum in order to study the stability, reproducibility, and development of the oral microbiome, and its dynamic response to environmental changes from the community to the molecular level.
Comparative metagenomic analyses confirmed a high similarity of metabolic potential in biofilms to recently available oral metagenomes from healthy subjects as part of the Human Microbiome Project. A time-series metagenomic analysis of the taxonomic community composition in biofilms revealed that the proportions of major species at 3 hours of growth are maintained during 48 hours of biofilm development. By employing deep pyrosequencing of the 16S rRNA gene to investigate this biofilm model with regards to bacterial taxonomic diversity, we show a high reproducibility of the taxonomic carriage and proportions between: 1) individual biofilm samples; 2) biofilm batches grown at different dates; 3) DNA extraction techniques and 4) research laboratories.
Our study demonstrates that we now have the capability to grow stable oral microbial in vitro biofilms containing more than one hundred operational taxonomic units (OTU) which represent 60-80% of the original inoculum OTU richness. Previously uncultivated Human Oral Taxa (HOT) were identified in the biofilms and contributed to approximately one-third of the totally captured 16S rRNA gene diversity. To our knowledge, this represents the highest oral bacterial diversity reported for an in vitro model system so far. This robust model will help investigate currently uncultivated species and the known virulence properties for many oral pathogens not solely restricted to pure culture systems, but within multi-species biofilms.
In vitro model; Biofilm; Oral microbiome; Saliva; Streptococcus; Lactobacillus; Uncultivated bacteria
The Clp/HSP100 family of molecular chaperones is ubiquitous in both prokaryotes and eukaryotes. These proteins play important roles in refolding, disaggregating and degrading proteins damaged by stress. As a subclass of the Clp/HSP100 family, ClpB has been shown to be involved in various stress responses as well as other functions in bacteria. In the present study, we investigated the role of a predicted ClpB-encoding gene, MXAN5092, in the stress response during vegetative growth and development of Myxococcus xanthus. Transcriptional analysis confirmed induction of this clpB homologue under different stress conditions, and further phenotypic analysis revealed that an in-frame deletion mutant of MXAN5092 was more sensitive to various stress treatments than the wild-type strain during vegetative growth. Moreover, the absence of the MXAN5092 gene resulted in decreased heat tolerance of myxospores, indicating the involvement of this clpB homologue in the stress response during the development of myxospores. The M. xanthus recombinant ClpB (MXAN5092) protein also showed a general chaperone activity in vitro. Overall, our genetic and phenotypic analysis of the predicted ATP-dependent chaperone protein ClpB (MXAN5092) demonstrated that it functions as a chaperone protein and plays an important role in cellular stress tolerance during both vegetative growth and development of M. xanthus.
The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy.
Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.
Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, direct observations of these interactions under native condition have not yet been made. In this study, we found that a truncated PilA protein (PilACt) which only contains the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein was constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin (WGA)-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able to efficiently label the EPS of M. xanthus and for the first time provided evidence for the direct interaction between the PilA protein and EPS under native conditions.
Type IV Pilin; Exopolysaccharides; Biofilm; Fruiting body; Confocal laser scanning microscopy; eGFP
One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA) within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM). Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS) in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.
As part of the human gastrointestinal tract, the oral cavity represents a complex biological system and harbors diverse bacterial species. Unlike the gut microbiota which is often considered a health asset, studies of the oral commensal microbial flora have been largely limited to their implication in oral diseases such as dental caries and periodontal diseases; Little emphasis has been given to their potential beneficial roles, especially the protective effects against oral colonization by foreign/pathogenic bacteria. In this study, we used the salivary microbiota derived from healthy human subjects to investigate protective effects against the colonization and integration of Pseudomonas aeruginosa, an opportunistic bacterial pathogen, into developing and pre-formed salivary biofilms. When co-cultivated in saliva medium, P. aeruginosa persisted in the planktonic phase, but failed to integrate into salivary microbial community during biofilm formation. Furthermore, in the saliva medium supplemented with 0.05% (w/v) sucrose, the oral flora inhibited the growth of P. aeruginosa by producing lactic acid. More interestingly, while pre-formed salivary biofilms were able to prevent P. aeruginosa colonization, the same biofilms recovered from mild chlorhexidine gluconate treatment displayed a shift in microbial composition and showed a drastic reduction in protection. Our study indicates that normal oral communities with balanced microbial compositions could be important in effectively preventing the integration of foreign/pathogenic bacterial species, such as P. aeruginosa.
bacterial interference; microbial flora; oral cavity; Pseudomonas aeruginosa; salivary biofilm