Dataset Information

Insertional inactivation of an intrageneric coaggregation-relevant adhesin locus from Streptococcus gordonii DL1 (Challis).

ABSTRACT: Transposon Tn916 was used to insertionally inactivate a coaggregation-relevant locus of Streptococcus gordonii DL1 (Challis). One mutant (F11) was isolated that lost the ability to coaggregate with the streptococcal partners of DL1 but retained the ability to coaggregate with partners belonging to other genera. A probe specific for the region flanking the Tn916 insertion was used to isolate a locus-specific fragment from a chromosomal lambda library. Southern analysis of the resulting phagemids revealed that a 0.5-kb EcoRI fragment hybridized with the F11 probe. Cloning of the 0.5-kb EcoRI fragment into the E. coli-streptococcal insertion vector p(omega) yielded pCW4, which was used to insertionally inactivate the putative coaggregation-relevant gene in DL1. Insertion mutants showed altered coaggregation with streptococci but retained wild-type coaggregation properties with other genera of bacteria. Comparison of immunoblots of cell surface proteins showed a 100-kDa protein in DL1 which was not detected in the Tn916 and pCW4 insertion mutants. These results indicate that the 0.5-kb EcoRI fragment is part of an adhesin-relevant locus that is involved in the production of a 100-kDa protein at the cell surface.

SUBMITTER: Whittaker CJ

PROVIDER: S-EPMC174348 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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Insertional inactivation of an intrageneric coaggregation-relevant adhesin locus from Streptococcus gordonii DL1 (Challis).

Whittaker C J CJ Clemans D L DL Kolenbrander P E PE

Infection and immunity 19961001 10

Transposon Tn916 was used to insertionally inactivate a coaggregation-relevant locus of Streptococcus gordonii DL1 (Challis). One mutant (F11) was isolated that lost the ability to coaggregate with the streptococcal partners of DL1 but retained the ability to coaggregate with partners belonging to other genera. A probe specific for the region flanking the Tn916 insertion was used to isolate a locus-specific fragment from a chromosomal lambda library. Southern analysis of the resulting phagemids ...[more]

PMID: 8926080

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Project description:Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded D-alanine-D-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of D-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137-4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that D-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.

Project description:Human oral viridans group streptococci that coaggregate with Actinomyces naeslundii PK606 express surface proteins related to ScaA, the coaggregation-mediating adhesin of Streptococcus gordonii PK488 (R. N. Andersen, N. Ganeshkumar, and P. E. Kolenbrander, Infect. Immun. 61:981-987, 1993). The nucleotide sequence of the 6,125-bp EcoRI insert of pRA1, containing scaA, the gene encoding ScaA, was determined. Six open reading frames (ORFs) were identified. The orientation of four ORFs, two upstream (ORF 1 and ORF 2) and one downstream (ORF 4) of scaA (ORF 3), indicated transcription in one direction, whereas ORF 5 and ORF 6 were transcribed divergently. Computer analysis of the deduced amino acid sequences identified a consensus binding site for ATP (GxxGxGKS) in the putative 28,054-Da protein encoded by ORF 1. ORF 2 potentially encoded a hydrophobic protein of 29,705 Da with six potential membrane-spanning regions. ScaA was 310 amino acids, 34,787 Da, and contained the lipoprotein consensus sequence LxxC, also reported for the ScaA-related proteins SsaB, FimA, and PsaA from Streptococcus sanguis 12, Streptococcus parasanguis FW213, and Streptococcus pneumoniae R36A, respectively. ORF 4 potentially encoded a 163-amino-acid protein of 17,912 Da, which was nearly identical to the downstream adjacent gene products of ssaB, fimA, and psaA. No significant homology with other proteins was found with the putative ORF 5 gene product, a 229-amino-acid protein of 25,107 Da. ORF 6 was incomplete and encoded a protein larger than 564 amino acids. This putative protein had a consensus Zn2+ binding motif, HExxH, found among bacterial thermolysins and mammalian neutral endopeptidases and was 40% identical to a homologous 210-amino-acid region of human enkephalinase. The genetic organization of ORFs 1, 2, and 3 was similar to those of the bacterial periplasmic-binding protein-dependent transport systems of gram-negative bacteria and binding-lipoprotein-dependent transport systems of gram-positive bacteria, and these genes appeared to encode ABC (ATP-binding cassette) proteins. This report describes a cell-to-cell adherence function associated with an ATP-binding cassette.

Project description:Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN, or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each species of bacteria when it was cultured alone. Likewise, cocultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different from the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernatants. Differing from what was found in a previous report, growth of S. mutans alone with GlcN inhibited the expression of multiple operons required for mutacin production. Cocultivation with S. gordonii consistently increased the expression of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes in S. mutans Conversely, S. gordonii appeared to be less affected by the presence of S. mutans but did show increases in genes for biosynthetic processes in the cocultures. In conclusion, amino sugars profoundly alter the interactions between pathogenic and commensal streptococci by reprogramming central metabolism.IMPORTANCE Carbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii), in an RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

Project description:Cell-cell adhesion between oral bacteria plays a key role in the development of polymicrobial communities such as dental plaque. Oral streptococci such as Streptococcus gordonii and Streptococcus oralis are important early colonizers of dental plaque and bind to a wide range of different oral microorganisms, forming multispecies clumps or "coaggregates." S. gordonii actively responds to coaggregation by regulating gene expression. To further understand these responses, we assessed gene regulation in S. gordonii and S. oralis following coaggregation in 25% human saliva. Coaggregates were formed by mixing, and after 30 min, RNA was extracted for dual transcriptome sequencing (RNA-Seq) analysis. In S. oralis, 18 genes (6 upregulated and 12 downregulated) were regulated by coaggregation. Significantly downregulated genes encoded functions such as amino acid and antibiotic biosynthesis, ribosome, and central carbon metabolism. In total, 28 genes were differentially regulated in Streptococcus gordonii (25 upregulated and 3 downregulated). Many genes associated with transporters and a two-component (NisK/SpaK) regulatory system were upregulated following coaggregation. Our comparative analyses of S. gordonii-S. oralis with different previously published S. gordonii pairings (S. gordonii-Fusobacterium nucleatum and S. gordonii-Veillonella parvula) suggest that the gene regulation is specific to each pairing, and responses do not appear to be conserved. This ability to distinguish between neighboring bacteria may be important for S. gordonii to adapt appropriately during the development of complex biofilms such as dental plaque. IMPORTANCE Dental plaque is responsible for two of the most prevalent diseases in humans, dental caries and periodontitis. Controlling the formation of dental plaque and preventing the transition from oral health to disease requires a detailed understanding of microbial colonization and biofilm development. Streptococci are among the most common colonizers of dental plaque. This study identifies key genes that are regulated when oral streptococci bind to one another, as they do in the early stages of dental plaque formation. We show that specific genes are regulated in two different oral streptococci following the formation of mixed-species aggregates. The specific responses of S. gordonii to coaggregation with S. oralis are different from those to coaggregation with other oral bacteria. Targeting the key genes that are upregulated during interspecies interactions may be a powerful approach to control the development of biofilm and maintain oral health.

Dataset Information

Insertional inactivation of an intrageneric coaggregation-relevant adhesin locus from Streptococcus gordonii DL1 (Challis).

Publications

Insertional inactivation of an intrageneric coaggregation-relevant adhesin locus from Streptococcus gordonii DL1 (Challis).

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