New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917.

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.     

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.     

Divergent structure of the ComQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis

In Bacillus subtilis, the ComQXPA quorum-sensing system controls cell density-dependent phenotypes such as the production of degradative enzymes and antibiotics and the development of genetic competence. Bacillus subtilis (natto) NAF12, a mutant defective in poly-gamma-glutamate (gamma-PGA) production, was derived from B. subtilis (natto) NAF4 by Tn917-LTV1 insertional mutagenesis. Determination of the mutant DNA sequences flanking the Tn917-LTV1 insert revealed that the insertion had inactivated comP in this mutant, indicating that gamma-PGA synthesis in B. subtilis (natto) is under the control of the ComP-ComA signal transduction system. A comparison of the amino acid sequences revealed striking variation in the primary structures of ComQ (44% identity), ComX (26%) and the sensor domain of ComP (36%) between B. subtilis (natto) NAF4 and B. subtilis 168. In contrast, the amino acid and nucleotide sequences of the kinase domains of ComP and of the ComA response regulator share 95% and 100% identity respectively. The comP genes of NAF4 and 168 restored the impaired competence of B. subtilis BD1658 (comP:cat) and gamma-PGA production of B. subtilis (natto) NAF12 (comP:Tn917-LTV1) to only 15% of the level achieved by the respective parent comP genes. However, when introduced together with the cognate comQ and comX genes, the comP genes restored the relevant defect of the heterologous comP mutants nearly to wild-type levels. Analogous to the comCDE system of Streptococcus strains and the agrBCDE system of Staphylococcus aureus, the concerted variation in the comQXP genes appears to establish specific intercellular communication between B. subtilis strains sharing the same pheromone system.     

Identification and characterization of genes controlled by the sporulation-regulatory gene spo0H in Bacillus subtilis.

We describe a general strategy for the identification of genes that are controlled by a specific regulatory factor in vivo and the use of this strategy to identify genes in Bacillus subtilis that are controlled by spo0H, a regulatory gene required for the initiation of sporulation. The general strategy makes use of a cloned regulatory gene fused to an inducible promoter to control expression of the regulatory gene and random gene fusions to a reporter gene to monitor expression in the presence and absence of the regulatory gene product. spo0H encodes a sigma factor of RNA polymerase, sigma H, and is required for the extensive reprograming of gene expression during the transition from growth to stationary phase and during the initiation of sporulation. We identified 18 genes that are controlled by sigma H (csh genes) in vivo by monitoring expression of random gene fusions to lacZ, made by insertion mutagenesis with the transposon Tn917lac, in the presence and absence of sigma H. These genes had lower levels of expression in the absence of sigma H than in the presence of sigma H. Patterns of expression of the csh genes during growth and sporulation in wild-type and spo0H mutant cells indicated that other regulatory factors are probably involved in controlling expression of some of these genes. Three of the csh::Tn917lac insertion mutations caused noticeable phenotypes. One caused a defect in vegetative growth, but only in combination with a spo0H mutation. Two others caused a partial defect in sporulation. One of these also caused a defect in the development of genetic competence. Detailed characterization of some of the csh genes and their regulatory regions should help define the role of spo0H in the regulation of gene expression during the transition from growth to stationary phase and during the initiation of sporulation.     

Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus

A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.     

Identification of the actin and plasminogen binding regions of group B streptococcal phosphoglycerate kinase

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in sign ificantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.     

Isolation of Bacillus subtilis transformation-deficient mutants and mapping of competence genes

We have isolated and characterized 48 Bacillus subtilis competence-deficient mutants. The mutants, obtained by nitrosoguanidine mutagenesis or by insertional mutagenesis with transposon Tn917, had a reduced transformation frequency and a wild-type transduction frequency. The com mutations were mapped by PBS1 transduction and at least four new com genes have been identified. The mutants were also characterized for their capacity to bind and take up the transforming DNA.     

转座子Tn917在短小芽孢杆菌中的转座作用

Transposition Tn917 was introduced into Bacillus pumilus 289 by protoplast transformation with plasmid pTV32. The temperature-sensitive replication property of pTV32 was maintained in B. pumilus. Tn917 was transposed efficiently in B. pumilus with 4.8 x 10(-4) transposition rate. The yield of auxotrophs was about 0.65% in all insertional mutants. It indicated a prospects for the use of Tn917 as a tool for insertional mutagenesis and genetic manipulation in B. pumilus.     

Cloning and molecular analysis of genes affecting expression of binding substance, the recipient-encoded receptor(s) mediating mating aggregate formation in Enterococcus faecalis.

Transfer of the conjugative plasmid pCF10 in Enterococcus faecalis strains involves production of a plasmid-encoded aggregation substance on the surface of donor cells in response to stimulation by a pheromone secreted by recipient cells. Aggregation substance then facilitates attachment to recipient cells via a chromosomally encoded receptor, termed binding substance (BS). A BS mutant, strain INY3000, generated by random Tn916 insertions, was previously found to carry copies of the transposon at four unique sites (K. M. Trotter and G. M. Dunny, Plasmid 24:57-67, 1990). In the present study, DNA flanking the Tn916 insertions was used to complement the BS mutation of INY3000 following Tn916 excision from cloned chromosomal fragments. Complementation results showed that three of the four regions mutated in INY3000 play some role in BS expression. Tn5 mutagenesis and DNA sequence analysis of the complementing fragment from one of these regions indicated the presence of three genes (ebsA, ebsB, and ebsC) that affect BS expression. The ebsA and ebsB genes encode peptides likely to function in cell wall metabolism, whereas ebsC may encode a product that suppresses the function or expression of EbsB.     

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.     

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements.

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.     

Cloning and expression of genes encoding pheromone-inducible antigens of Enterococcus (Streptococcus) faecalis.

Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tra130 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118- to 125-kDa antigens (designated pre-Tra130) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, Tra150, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cloned EcoRI C and E fragments). The coding sequences for the Tra150 and Tra130 antigens were further localized in the TRA region of pCF10 by transposon insertion mutagenesis. Western blot analyses of the recombinant strains, and of strains carrying derivatives of pCF10 or various recombinant plasmids containing Tn5 or Tn917 insertions, suggested that the portion of pCF10 comprising the tra3 through -6 segments (previously defined by Tn917 insertional mutagenesis) contained several genes that are involved in regulating the synthesis of Tra130 and Tra150.