Natural genetic transformation in Streptococcus gordonii: comX imparts spontaneous competence on strain wicky.

Streptococcus gordonii Wicky becomes competent only after stimulation with conditioned medium from strain Challis as a source of competence factor (CF). A 3.2-kbp genomic fragment from Challis was found to impart spontaneous competence on Wicky by a complementation assay. Wicky clones containing the fragment secreted a heat-sensitive activity that induced competence in Wicky and in a comA insertion mutant of Challis. Activity was localized to a putative open reading frame, comX, with the potential to encode a 52-amino-acid peptide. comX had no similarity to known sequences, and a comX::ermAM insertion mutant of Challis transformed normally and secreted CF. These data suggest that a CF-independent pathway for competence induction exists in S. gordonii.     

Genome-wide transcriptional response of Yersinia pestis to stressful conditions simulating phagolysosomal environments

Yersinia pestis is a Gram-negative coccobacillus causing the dangerous disease, plague. Survival of Y. pestis within host macrophages is important in the initial stages of infection. In our present work, DNA microarray was used to determine the expression profiles of Y. pestis strain 201 in response to in vitro simulating conditions of Mg(2+) limitation, polymyxin treatment and oxidative stress that could be found in phagolysosomal environment. It was demonstrated that Y. pestis made appropriate adaptive/protective responses to survive the stressful environments. There are the induced expression of antiphagocytic factors and Mg(2+) transporters under Mg(2+) limitation condition, the stimulation of drug/analogue sensitivity and glycerol assimilation after polymyxin treatment, and the differential expression in genes encoding stress-responsive proteins, components of cell envelope, iron assimilation and regulatory functions in response to both Mg(2+) limitation and polymyxin treatment. Under oxidative stress, Y. pestis uses several mechanisms, especially including the induced expression of detoxification enzymes and DNA repair proteins, to protect from or repair the oxidative cell damages. This microarray analysis would provide the candidates for identifying genes or pathways required for growth and proliferation of Y. pestis in macrophages.     

Genome-wide transcriptional changes of ramie (Boehmeria nivea L. Gaud) in response to root-lesion nematode infection

Ramie fiber extracted from stem bark is one of the most important natural fibers. The root-lesion nematode (RLN) Pratylenchus coffeae is a major ramie pest and causes large fiber yield losses in China annually. The response mechanism of ramie to RLN infection is poorly understood. In this study, we identified genes that are potentially involved in the RLN-resistance in ramie using Illumina pair-end sequencing in two RLN-infected plants (Inf1 and Inf2) and two control plants (CO1 and CO2). Approximately 56.3, 51.7, 43.4, and 45.0 million sequencing reads were generated from the libraries of CO1, CO2, Inf1, and Inf2, respectively. De novo assembly for these 196 million reads yielded 50,486 unigenes with an average length of 853.3 bp. A total of 24,820 (49.2%) genes were annotated for their function. Comparison of gene expression levels between CO and Inf ramie revealed 777 differentially expressed genes (DEGs). The expression levels of 12 DEGs were further confirmed by real-time quantitative PCR (qRT-PCR). Pathway enrichment analysis showed that three pathways (phenylalanine metabolism, carotenoid biosynthesis, and phenylpropanoid biosynthesis) were strongly influenced by RLN infection. A series of candidate genes and pathways that may contribute to the defense response against RLN in ramie will be helpful for further improving resistance to RLN infection.     

Streptococcus gordonii Hsa environmentally constrains competitive binding by Streptococcus sanguinis to saliva-coated hydroxyapatite

Competition between pioneer colonizing bacteria may determine polymicrobial succession during dental plaque development, but the ecological constraints are poorly understood. For example, more Streptococcus sanguinis than Streptococcus gordonii organisms are consistently isolated from the same intraoral sites, yet S. gordonii fails to be excluded and survives as a species over time. To explain this observation, we hypothesized that S. gordonii could compete with S. sanguinis to adhere to saliva-coated hydroxyapatite (sHA), an in vitro model of the tooth surface. Both species bound similarly to sHA, yet 10- to 50-fold excess S. gordonii DL1 reduced binding of S. sanguinis SK36 by 85 to >95%. S. sanguinis, by contrast, did not significantly compete with S. gordonii to adhere. S. gordonii competed with S. sanguinis more effectively than other species of oral streptococci and depended upon the salivary film on HA. Next, putative S. gordonii adhesins were analyzed for contributions to interspecies competitive binding. Like wild-type S. gordonii, isogenic mutants with mutations in antigen I/II polypeptides (sspAB), amylase-binding proteins (abpAB), and Csh adhesins (cshAB) competed effectively against S. sanguinis. By contrast, an hsa-deficient mutant of S. gordonii showed significantly reduced binding and competitive capabilities, while these properties were restored in an hsa-complemented strain. Thus, Hsa confers a selective advantage to S. gordonii over S. sanguinis in competitive binding to sHA. Hsa expression may, therefore, serve as an environmental constraint against S. sanguinis, enabling S. gordonii to persist within the oral cavity, despite the greater natural prevalence of S. sanguinis in plaque and saliva.     

Expression of measles virus antigens in Streptococcus gordonii

The measles virus proteins haemagglutinin (HA) and fusion protein (F), which together mediate attachment and penetration of the virus in the host cell and can elicit production of neutralising antibodies in the course of natural infection were expressed in the vaccine vector Streptococcus gordonii, a Gram-positive bacterium normally present in the human oral cavity. HA and F were expressed as fusion proteins attached to the bacterial surface, and were both found to be immunogenic when the recombinant S. gordonii were inoculated subcutaneously in mice.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis

Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-beta-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.     

Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Streptococcus gordonii

Disulfide bonds are important for the stability of many extracellular proteins, including bacterial virulence factors. Formation of these bonds is catalyzed by thiol-disulfide oxidoreductases (TDORs). Little is known about their formation in Gram-positive bacteria, particularly among facultative anaerobic Firmicutes, such as streptococci. To investigate disulfide bond formation in Streptococcus gordonii, we identified five putative TDORs from the sequenced genome. Each of the putative TDOR genes was insertionally inactivated with an erythromycin resistance cassette, and the mutants were analyzed for autolysis, extracellular DNA release, biofilm formation, bacteriocin production, and genetic competence. This analysis revealed a single TDOR, SdbA, which exhibited a pleiotropic mutant phenotype. Using an in silico analysis approach, we identified the major autolysin AtlS as a natural substrate of SdbA and showed that SdbA is critical to the formation of a disulfide bond that is required for autolytic activity. Analysis by BLAST search revealed homologs to SdbA in other Gram-positive species. This study provides the first in vivo evidence of an oxidoreductase, SdbA, that affects multiple phenotypes in a Gram-positive bacterium. SdbA shows low sequence homology to previously identified oxidoreductases, suggesting that it may belong to a different class of enzymes. Our results demonstrate that SdbA is required for disulfide bond formation in S. gordonii and indicate that this enzyme may represent a novel type of oxidoreductase in Gram-positive bacteria.