Search tips
Search criteria

Results 1-25 (85)

Clipboard (0)

Select a Filter Below

Year of Publication
more »
author:("Shi, wenchuan")
1.  Characterization of aid1, a novel gene involved in Fusobacterium nucleatum interspecies interactions 
Microbial ecology  2014;68(2):379-387.
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.
PMCID: PMC4104215  PMID: 24643713
F. nucleatum; oral streptococci; interspecies interaction; RadD
2.  The social structure of microbial community involved in colonization resistance 
The ISME Journal  2013;8(3):564-574.
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.
PMCID: PMC3930314  PMID: 24088624
colonization resistance; microbial community; social structure; host-associated microbiota; facilitation
3.  Development of In Vitro Denture Biofilm Models for Halitosis Related Bacteria and their Application in Testing the Efficacy of Antimicrobial Agents 
The Open Dentistry Journal  2015;9:125-131.
Objective : 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.
PMCID: PMC4407000  PMID: 25926895
Antimicrobial treatment; biofilms; denture; halitosis; model
4.  Effects on Hardness and Elastic Modulus for DSS-8 Peptide Treatment on Remineralization of Human Dental Tissues 
Dental remineralization may be achieved by mediating the interactions between tooth surfaces with free ions and biomimetic peptides. We recently developed octuplet repeats of aspartate-serine-serine (DSS-8) peptide, which occurs in high abundance in naturally occurring proteins that are critical for tooth remineralization. In this paper, we evaluated the possible role of DSS-8 in enamel remineralization. Human enamel specimens were demineralized, exposed briefly to DSS-8 solution, and then exposed to concentrated ionic solutions that favor remineralization. Enamel nano-mechanical behaviors, hardness and elastic modulus, at various stages of treatment were determined by nanoindentation. The phase, microstructure and morphology of the resultant surfaces were characterized using the grazing incidence X-ray diffraction (GIXD), variable pressure scanning electron microscopy (VPSEM), and atomic force microscopy (AFM), respectively. Nanoindentation results show that the DSS-8 remineralization effectively improves the mechanical and elastic properties for demineralized enamel.
PMCID: PMC4209483  PMID: 25355990
Enamel; Peptide; Nanoindentation; Remineralization
5.  Killing of Escherichia coli by Myxococcus xanthus in Aqueous Environments Requires Exopolysaccharide-dependent Physical Contact 
Microbial ecology  2013;66(3):630-638.
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.
PMCID: PMC3931608  PMID: 23828520
M. xanthus; contact-dependent interaction; co-aggregation; aqueous environment
6.  Oral Microbiota Distinguishes Acute Lymphoblastic Leukemia Pediatric Hosts from Healthy Populations 
PLoS ONE  2014;9(7):e102116.
In leukemia, oral manifestations indicate aberrations in oral microbiota. Microbiota structure is determined by both host and environmental factors. In human hosts, how health status shapes the composition of oral microbiota is largely unknown. Taking advantage of advances in high-throughput sequencing, we compared the composition of supragingival plaque microbiota of acute lymphoblastic leukemia (ALL) pediatric patients with healthy controls. The oral microbiota of leukemia patients had lower richness and less diversity compared to healthy controls. Microbial samples clustered into two major groups, one of ALL patients and another of healthy children, with different structure and composition. Abundance changes of certain taxa including the Phylum Firmicutes, the Class Bacilli, the Order Lactobacillales, the Family Aerococcaceae and Carnobacteriaceae, as well as the Genus Abiotrophia and Granulicatella were associated with leukemia status. ALL patients demonstrated a structural imbalance of the oral microbiota, characterized by reduced diversity and abundance alterations, possibly involved in systemic infections, indicating the importance of immune status in shaping the structure of oral microbiota.
PMCID: PMC4099009  PMID: 25025462
7.  Intercellular communications in multispecies oral microbial communities 
The oral cavity contains more than 700 microbial species that are engaged in extensive cell–cell interactions. These interactions contribute to the formation of highly structured multispecies communities, allow them to perform physiological functions, and induce synergistic pathogenesis. Co-adhesion between oral microbial species influences their colonization of oral cavity and effectuates, to a large extent, the temporal and spatial formation of highly organized polymicrobial community architecture. Individual species also compete and collaborate with other neighboring species through metabolic interactions, which not only modify the local microenvironment such as pH and the amount of oxygen, making it more suitable for the growth of other species, but also provide a metabolic framework for the participating microorganisms by maximizing their potential to extract energy from limited substrates. Direct physical contact of bacterial species with its neighboring co-habitants within microbial community could initiate signaling cascade and achieve modulation of gene expression in accordance with different species it is in contact with. In addition to communication through cell–cell contact, quorum sensing (QS) mediated by small signaling molecules such as competence-stimulating peptides (CSPs) and autoinducer-2 (AI-2), plays essential roles in bacterial physiology and ecology. This review will summarize the evidence that oral microbes participate in intercellular communications with co-inhabitants through cell contact-dependent physical interactions, metabolic interdependencies, as well as coordinative signaling systems to establish and maintain balanced microbial communities.
PMCID: PMC4076886  PMID: 25071741
oral microbial community; coadhesion; signaling transduction; metabolic interactions; cell-cell communication
8.  Chromosomal DNA deletion confers phage resistance to Pseudomonas aeruginosa 
Scientific Reports  2014;4:4738.
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.
PMCID: PMC4001099  PMID: 24770387
9.  Development of HuMiChip for Functional Profiling of Human Microbiomes 
PLoS ONE  2014;9(3):e90546.
Understanding the diversity, composition, structure, function, and dynamics of human microbiomes in individual human hosts is crucial to reveal human-microbial interactions, especially for patients with microbially mediated disorders, but challenging due to the high diversity of the human microbiome. Here we have developed a functional gene-based microarray for profiling human microbiomes (HuMiChip) with 36,802 probes targeting 50,007 protein coding sequences for 139 key functional gene families. Computational evaluation suggested all probes included are highly specific to their target sequences. HuMiChip was used to analyze human oral and gut microbiomes, showing significantly different functional gene profiles between oral and gut microbiome. Obvious shifts of microbial functional structure and composition were observed for both patients with dental caries and periodontitis from moderate to advanced stages, suggesting a progressive change of microbial communities in response to the diseases. Consistent gene family profiles were observed by both HuMiChip and next generation sequencing technologies. Additionally, HuMiChip was able to detect gene families at as low as 0.001% relative abundance. The results indicate that the developed HuMiChip is a useful and effective tool for functional profiling of human microbiomes.
PMCID: PMC3942451  PMID: 24595026
10.  Transcriptional Responses of Treponema denticola to Other Oral Bacterial Species 
PLoS ONE  2014;9(2):e88361.
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.
PMCID: PMC3914990  PMID: 24505483
11.  Salivary Biomarkers for Caries Risk Assessment 
Saliva contains various microbes and host biological components that could be used for caries risk assessment. This review focuses on the research topics that connect dental caries with saliva, including both the microbial and host components within saliva.
PMCID: PMC3825179  PMID: 23505756
12.  Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans 
Molecular oral microbiology  2012;28(2):10.1111/omi.12014.
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.
PMCID: PMC3819222  PMID: 23241367
foldase protein PrsA; protein secretion; Streptococcus mutans
13.  Effects of Exopolysaccharide Production on Liquid Vegetative Growth, Stress Survival and Stationary Phase Recovery in Myxococcus xanthus 
Journal of microbiology (Seoul, Korea)  2012;50(2):10.1007/s12275-012-1349-5.
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.
PMCID: PMC3819231  PMID: 22538652
Myxococcus xanthus; exopolysaccharide; vegetative growth; stress survival; stationary phase recovery
14.  An in vitro biofilm model system maintaining a highly reproducible species and metabolic diversity approaching that of the human oral microbiome 
Microbiome  2013;1:25.
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.
PMCID: PMC3971625  PMID: 24451062
In vitro model; Biofilm; Oral microbiome; Saliva; Streptococcus; Lactobacillus; Uncultivated bacteria
15.  The clpB gene is involved in the stress response of Myxococcus xanthus during vegetative growth and development 
Microbiology  2012;158(Pt 9):2336-2343.
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.
PMCID: PMC3542817  PMID: 22790397
16.  Mapping the Tail Fiber as the Receptor Binding Protein Responsible for Differential Host Specificity of Pseudomonas aeruginosa Bacteriophages PaP1 and JG004 
PLoS ONE  2013;8(7):e68562.
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.
PMCID: PMC3706319  PMID: 23874674
17.  Acyl homoserine lactone-based quorum sensing in a methanogenic archaeon 
The ISME Journal  2012;6(7):1336-1344.
Acyl homoserine lactone (AHL)-based quorum sensing commonly refers to cell density-dependent regulatory mechanisms found in bacteria. However, beyond bacteria, this cell-to-cell communication mechanism is poorly understood. Here we show that a methanogenic archaeon, Methanosaeta harundinacea 6Ac, encodes an active quorum sensing system that is used to regulate cell assembly and carbon metabolic flux. The methanogen 6Ac showed a cell density-dependent physiology transition, which was related to the AHL present in the spent culture and the filI gene-encoded AHL synthase. Through extensive chemical analyses, a new class of carboxylated AHLs synthesized by FilI protein was identified. These carboxylated AHLs facilitated the transition from a short cell to filamentous growth, with an altered carbon metabolic flux that favoured the conversion of acetate to methane and a reduced yield in cellular biomass. The transcriptomes of the filaments and the short cell forms differed with gene expression profiles consistent with the physiology. In the filaments, genes encoding the initial enzymes in the methanogenesis pathway were upregulated, whereas those for cellular carbon assimilation were downregulated. A luxI–luxR ortholog filI–filR was present in the genome of strain 6Ac. The carboxylated AHLs were also detected in other methanogen cultures and putative filI orthologs were identified in other methanogenic genomes as well. This discovery of AHL-based quorum sensing systems in methanogenic archaea implies that quorum sensing mechanisms are universal among prokaryotes.
PMCID: PMC3379639  PMID: 22237544
carboxylated acyl homoserine lactones; filI-encoded AHL synthase; methanogenic archaea; physiology transition; quorum sensing
18.  Investigating Acid Production by Streptococcus mutans with a Surface-Displayed pH-Sensitive Green Fluorescent Protein 
PLoS ONE  2013;8(2):e57182.
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.
PMCID: PMC3585301  PMID: 23468929
19.  Direct visualization of the interaction between pilin and exopolysaccharides of Myxococcus xanthus with eGFP fused PilA protein 
FEMS microbiology letters  2011;326(1):23-30.
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.
PMCID: PMC3454480  PMID: 22092602
Type IV Pilin; Exopolysaccharides; Biofilm; Fruiting body; Confocal laser scanning microscopy; eGFP
20.  DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides 
PLoS ONE  2012;7(12):e51905.
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.
PMCID: PMC3530553  PMID: 23300576
21.  Community-based interference against integration of Pseudomonas aeruginosa into human salivary microbial biofilm 
Molecular Oral Microbiology  2011;26(6):337-352.
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.
PMCID: PMC3327514  PMID: 22053962
bacterial interference; microbial flora; oral cavity; Pseudomonas aeruginosa; salivary biofilm
22.  Evaluation of bacteria-induced enamel demineralization using optical profilometry 
Streptococcus mutans is considered a major causative of tooth decay due to it’s ability to rapidly metabolize carbohydrates such as sucrose. One prominent excreted end product of sucrose metabolism is lactic acid. Lactic acid causes a decrease in the pH of the oral environment with subsequent demineralization of the tooth enamel. Biologically relevant bacteria-induced enamel demineralization was studied.
Optical profiling was used to measure tooth enamel decay with vertical resolution under one nanometer and lateral features with optical resolution as a result of S. mutans biofilm exposure. Comparison measurements were made using AFM.
After 72 hr of biofilm exposure the enamel displayed an 8-fold increase in the observed roughness average, (Ra), as calculated over the entire measured array. Similarly, the average root mean square (RMS) roughness, RRMS, of the enamel before and after biofilm exposure for 3 days displayed a 7-fold increase. Further, the direct effect of chemically induced enamel demineralization using biologically relevant organic acids was shown. Optical profiles of the enamel surface after addition of a 30% lactic acid solution showed a significant alteration in the surface topography with a corresponding increase in respective surface roughness statistics. Similar measurements with 10% citric acid over seconds and minutes give insight into the demineralization process by providing quantitative measures for erosion rates: comparing surface height and roughness as metrics.
The strengths of optical profilometry as an analytical tool for understanding and analyzing biologically relevant processes such as biofilm induced tooth enamel demineralization were demonstrated.
PMCID: PMC3454478  PMID: 19732947
enamel erosion; optical profilometry; biofilm; Streptococcus mutans; enamel demineralization; citric acid; lactic acid; AFM
23.  Oligomerization of the Response Regulator ComE from Streptococcus mutans Is Affected by Phosphorylation 
Journal of Bacteriology  2012;194(5):1127-1135.
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.
PMCID: PMC3294772  PMID: 22210762
24.  Molecular Characterization of the Microbiota Residing at the Apical Portion of Infected Root Canals of Human Teeth 
Journal of endodontics  2011;37(10):1359-1364.
This study investigated the bacterial communities residing in the apical portion of human teeth with apical periodontitis in primary and secondary infections using a culture-independent molecular biology approach.
Root canal samples from the apical root segments of extracted teeth were collected from 18 teeth with necrotic pulp and 8 teeth with previous endodontic treatment. Samples were processed for amplification via polymerase chain reaction (PCR) and separated with denaturing gradient gel electrophoresis (DGGE). Selected bands were excised from the gel and sequenced for identification.
Comparable to previous studies of entire root canals, the apical bacterial communities in primary infections were significantly more diverse than in secondary infections (p=0.0003). Inter- and intra-patient comparisons exhibited similar variations in profiles. Different roots of the same teeth with secondary infections displayed low similarity in bacterial composition, while an equivalent sample collected from primary infection contained almost identical populations. Sequencing revealed a high prevalence of fusobacteria, Actinomyces sp. and oral Anaeroglobus geminatus in both types of infection. Many secondary infections contained Burkholderiales or Pseudomonas sp. both of which represent opportunistic environmental pathogens.
Certain microorganisms exhibit similar prevalence in primary and secondary infection indicating that they are likely not eradicated during endodontic treatment. The presence of Burkholderiales and Pseudomonas sp. underscores the problem of environmental contamination. Treatment appears to affect the various root canals of multi-rooted teeth differently, resulting in local changes of the microbiota.
PMCID: PMC3415298  PMID: 21924182
Apical periodontitis; endodontic infections; community profiling; polymerase chain reaction; denaturing gradient gel electrophoresis
25.  Alanine 32 in PilA is important for PilA stability and type IV pili function in Myxococcus xanthus 
Microbiology  2011;157(Pt 7):1920-1928.
Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure–function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.
PMCID: PMC3167889  PMID: 21493683

Results 1-25 (85)