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author:("Shi, wenchuan")
1.  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
2.  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
3.  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
4.  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
5.  Adherence to Streptococci facilitates Fusobacterium nucleatum integration into an oral microbial community 
Microbial Ecology  2011;63(3):532-542.
The development of multispecies oral microbial communities involves complex intra- and interspecies interactions at various levels. The ability to adhere to the resident bacteria or the biofilm matrix and overcome community resistance are among the key factors that determine whether a bacterium can integrate into a community. In this study, we focus on community integration of Fusobacterium nucleatum, a prevalent Gram-negative oral bacterial species that is considered an important member of the oral community due to its ability to adhere to Gram-positive as well as Gram-negative species. This interaction with a variety of different species is thought to facilitate the establishment of multispecies oral microbial community. However, the majority of experiments thus far has focused on the physical adherence between two species as measured by in vitro co-aggregation assays, while the community-based effects on the integration of F. nucleatum into multispecies microbial community remains to be investigated. In this study, we demonstrated using an established in vitro mice oral microbiota (O-mix) that the viability of F. nucleatum was significantly reduced upon addition to the O-mix due to cell contact-dependent induction of hydrogen peroxide (H2O2) production by oral community. Interestingly, this inhibitory effect was significantly alleviated when F. nucleatum was allowed to adhere to its known interacting partner species (such as Streptococcus sanguinis) prior to addition. Furthermore, this aggregate formation-dependent protection was absent in the F. nucleatum mutant strain ΔFn1526 that is unable to bind to a number of Gram-positive species. More importantly, this protective effect was also observed during integration of F. nucleatum into a human salivary microbial community (S-mix). These results support the idea that by adhering to other oral microbes, such as streptococci, F. nucleatum is able to mask the surface components that are recognized by H2O2 producing oral community members. This evasion strategy prevents detection by antagonistic oral bacteria and allows integration into the developing oral microbial community.
PMCID: PMC3313671  PMID: 22202886
coaggregation; Fusobacterium nucleatum; microbial flora; oral cavity; community resistance
6.  Selective Membrane Disruption: Mode of Action of C16G2, a Specifically Targeted Antimicrobial Peptide ▿ 
The specifically targeted antimicrobial peptide (STAMP) C16G2 was developed to target the cariogenic oral pathogen Streptococcus mutans. Because the design of this peptide was novel, we sought to better understand the mechanism through which it functioned. Compared to antimicrobial peptides (AMPs) with wide spectra of activity, the STAMP C16G2 has demonstrated specificity for S. mutans in a mixed-culture environment, resulting in the complete killing of S. mutans while having minimal effect on the other streptococci. In the current study, we sought to further confirm the selectivity of C16G2 and also compare its membrane activity to that of melittin B, a classical toxic AMP, in order to determine the STAMP's mechanism of cell killing. Disruption of S. mutans cell membranes by C16G2 was demonstrated by increased SYTOX green uptake and ATP efflux from the cells similar to those of melittin B. Treatment with C16G2 also resulted in a loss of membrane potential as measured by DiSC(3)5 fluorescence. In comparison, the individual moieties of C16G2 demonstrated no specificity and limited antimicrobial activity compared to those of the STAMP C16G2. The data suggest that C16G2 has a mechanism of action similar to that of traditional AMPs and kills S. mutans through disruption of the cell membrane, allowing small molecules to leak out of the cell, which is followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans, but not other noncariogenic oral streptococci.
PMCID: PMC3122425  PMID: 21518845
7.  Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans▿† 
Journal of Bacteriology  2011;193(14):3642-3652.
In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.
PMCID: PMC3133340  PMID: 21602345
8.  Natural Transformation of Myxococcus xanthus▿† 
Journal of Bacteriology  2011;193(9):2122-2132.
Myxococcus xanthus belongs to the delta class of the proteobacteria and is notable for its complex life-style with social behaviors and relatively large genome. Although previous observations have suggested the existence of horizontal gene transfer in M. xanthus, its ability to take up exogenous DNA via natural transformation has not been experimentally demonstrated. In this study, we achieved natural transformation in M. xanthus using the autonomously replicating myxobacterial plasmid pZJY41 as donor DNA. M. xanthus exopolysaccharide (EPS) was shown to be an extracellular barrier for transformation. Cells deficient in EPS production, e.g., mutant strains carrying ΔdifA or ΔepsA, became naturally transformable. Among the inner barriers to transformation were restriction-modification systems in M. xanthus, which could be partially overcome by methylating DNA in vitro using cell extracts of M. xanthus prior to transformation. In addition, the incubation time of DNA with cells and the presence of divalent magnesium ion affected transformation frequency of M. xanthus. Furthermore, we also observed a potential involvement of the type IV pilus system in the DNA uptake machinery of M. xanthus. The natural transformation was totally eliminated in the ΔpilQ/epsA and Δtgl/epsA mutants, and null mutation of pilB or pilC in an ΔepsA background diminished the transformation rate. Our study, to the best of our knowledge, provides the first example of a naturally transformable species among deltaproteobacteria.
PMCID: PMC3133062  PMID: 21378184
9.  Rapid Probing of Biological Surfaces with a Sparse-Matrix Peptide Library 
PLoS ONE  2011;6(8):e23551.
Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained “hits” against all biological surfaces probed. Sequence refinement of the “hits” led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue.
PMCID: PMC3156232  PMID: 21858167
10.  Experimentally Guided Computational Model Discovers Important Elements for Social Behavior in Myxobacteria 
PLoS ONE  2011;6(7):e22169.
Identifying essential factors in cellular interactions and organized movement of cells is important in predicting behavioral phenotypes exhibited by many bacterial cells. We chose to study Myxococcus xanthus, a soil bacterium whose individual cell behavior changes while in groups, leading to spontaneous formation of aggregation center during the early stage of fruiting body development. In this paper, we develop a cell-based computational model that solely relies on experimentally determined parameters to investigate minimal elements required to produce the observed social behaviors in M. xanthus. The model verifies previously known essential parameters and identifies one novel parameter, the active turning, which we define as the ability and tendency of a cell to turn to a certain angle without the presence of any obvious external factors. The simulation is able to produce both gliding pattern and spontaneous aggregation center formation as observed in experiments. The model is tested against several known M. xanthus mutants and our modification of parameter values relevant for the individual mutants produces good phenotypic agreements. This outcome indicates the strong predictive potential of our model for the social behaviors of uncharacterized mutants and their expected phenotypes during development.
PMCID: PMC3139613  PMID: 21811570
11.  Exopolysaccharide-Independent Social Motility of Myxococcus xanthus 
PLoS ONE  2011;6(1):e16102.
Social motility (S motility), the coordinated movement of large cell groups on agar surfaces, of Myxococcus xanthus requires type IV pili (TFP) and exopolysaccharides (EPS). Previous models proposed that this behavior, which only occurred within cell groups, requires cycles of TFP extension and retraction triggered by the close interaction of TFP with EPS. However, the curious observation that M. xanthus can perform TFP-dependent motility at a single-cell level when placed onto polystyrene surfaces in a highly viscous medium containing 1% methylcellulose indicated that “S motility” is not limited to group movements. In an apparent further challenge of the previous findings for S motility, mutants defective in EPS production were found to perform TFP-dependent motility on polystyrene surface in methylcellulose-containing medium. By exploring the interactions between pilin and surface materials, we found that the binding of TFP onto polystyrene surfaces eliminated the requirement for EPS in EPS- cells and thus enabled TFP-dependent motility on a single cell level. However, the presence of a general anchoring surface in a viscous environment could not substitute for the role of cell surface EPS in group movement. Furthermore, EPS was found to serve as a self-produced anchoring substrate that can be shed onto surfaces to enable cells to conduct TFP-dependent motility regardless of surface properties. These results suggested that in certain environments, such as in methylcellulose solution, the cells could bypass the need for EPS to anchor their TPF and conduct single-cell S motility to promote exploratory movement of colonies over new specific surfaces.
PMCID: PMC3016331  PMID: 21245931

Results 1-11 (11)