Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes

Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.     

L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein.

The intracellular pathogenic bacterium L. monocytogenes can spread directly from cell to cell without leaving the cytoplasm. The mechanism of this movement, generated through bacterially induced actin polymerization, is not understood. By analyzing an avirulent Tn917-lac mutant defective for actin polymerization, we have identified a bacterial component involved in this process. The transposon had inserted in actA, the second gene of an operon. Gene disruption of downstream genes and transformation of the mutant strain with actA showed that the actA gene encodes a surface protein necessary for bacterially induced actin assembly. Our results indicate that it is a 610 amino acid protein with an apparent molecular weight of 90 kd.     

The Myxococcus xanthus wbgB gene encodes a glycosyltransferase homologue required for lipopolysaccharide O-antigen biosynthesis

Myxococcus xanthus is a gram-negative soil bacterium that initiates a complex developmental program in response to starvation. A transposon insertion (Tn5-lac omega109) mutant with developmental deficiencies was isolated and characterized in this study. A strain containing this insertion mutation in an otherwise wild-type background showed delayed developmental aggregation for about 12 h and sporulated at 1-2% of the wild-type level. Tn5-lac omega109 was found to have disrupted the M. xanthus wbgB gene, which is located 2.1 kb downstream of the M. xanthus lipopolysacharide (LPS) O-antigen biosynthesis genes wzm wzt wbgA. The deduced polypeptide sequence of WbgB shares significant similarity with bacterial glycosyltransferases including M. xanthus WbgA. The wbgB::Tn5-lac omega109 mutant was found to be defective in LPS O-antigen synthesis by immunochemical analysis. Further mutational analysis indicated that the defects of the wbgB::Tn5-lac omega109 mutant were not the result of polar effects on downstream genes. Various motility assays demonstrated that the Tn5-lac omega109 mutation affected both social (S) and adventurous (A) gliding motility of M. xanthus cells. The pleiotrophic effects of wbgB mutations indicate the importance of LPS O-antigen biosynthesis for various cellular functions in M. xanthus.     

Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.     

The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein.

Expression of beta-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M.R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrgAB. The nrgA gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli glnB-encoded PII protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis sigma A-dependent promoters.     

Delivery system for creation of one-step in vivo lac gene fusions in Pseudomonas spp. involved in biological control

The suicide plasmid pVA838 carrying the operon fusion transposon Tn5-lac was used as a delivery system to introduce Tn5-lac into Pseudomonas sp. strain M114. Random, in vivo lac gene fusions were successfully isolated in a one-step conjugation approach with this vector system. Tn5-lac-containing exconjugants were recovered at a frequency of approximately 10(-7) per recipient. However, when the mating temperature was increased from the normal growth temperature (28 degrees C) to 34 degrees C, the frequency was increased to approximately 10(-4) per recipient. A number of in vivo lac gene fusions were isolated and characterized in strain M114, a potentially important bacterium for biological control purposes.     

Cloning and sequencing of the gerD gene of Bacillus subtilis

A Tn917 insertion in the same region of the chromosome as gerD gave rise to a mutant (ger-97) with a germination phenotype similar to that of two gerD mutants which germinate abnormally in a range of germinants. The insertion and two gerD mutations were cotransformed with ribosomal protein genes rpoB, rpsE and rpsI. DNA cloned from one side of the insertion carried the 16S end of the ribosomal RNA operon rrnI. These data were consistent with the order rpoB-rpsE-rpsI-gerD/ger-97::Tn917-rrnI. Insertion into the wild-type chromosome of a plasmid carrying DNA adjacent to the insertion permitted the recovery of a 1.8 kb fragment of DNA which complemented ger-97::Tn917 and the gerD mutations. The DNA nucleotide sequence of the region of this fragment at which Tn917 had inserted revealed a 555 bp open reading frame, preceded by a ribosome-binding site and potential sigma E and sigma A promoter regions and encoding a predicted polypeptide of 21,117 Da. This polypeptide was largely hydrophilic but contained a hydrophobic region at the N-terminus resembling a signal peptide.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Delivery system for creation of one-step in vivo lac gene fusions in Pseudomonas spp. involved in biological control.

The suicide plasmid pVA838 carrying the operon fusion transposon Tn5-lac was used as a delivery system to introduce Tn5-lac into Pseudomonas sp. strain M114. Random, in vivo lac gene fusions were successfully isolated in a one-step conjugation approach with this vector system. Tn5-lac-containing exconjugants were recovered at a frequency of approximately 10(-7) per recipient. However, when the mating temperature was increased from the normal growth temperature (28 degrees C) to 34 degrees C, the frequency was increased to approximately 10(-4) per recipient. A number of in vivo lac gene fusions were isolated and characterized in strain M114, a potentially important bacterium for biological control purposes.     

Gene-specific transposon mutagenesis of the biphenyl/polychlorinated biphenyl-degradation-controlling bph operon in soil bacteria.

A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.