Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa. The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5' of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria. This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa. Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD+ dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5' from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to alpha-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.     

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.     

Efficient insertional mutagenesis in group A streptococci mediated by Tn917 transposon

The replication-conditional thermosensitive vector pTV1-OK (repATs, Kan^r) harbouring the transposon Tn917 (Em^r) was successfully used to mutagenise a clinical Streptococcus pyogenes isolate (CS101). In the initial studies, conditions were established for electrotransformation of the pTV1-OK vector into CS101. Transformants selected on media containing Kan at 29°C were shown to become Kan^s at 39°C and to carry the transposon-linked Em^r marker. One such transformant was chosen for transposition studies. Upon temperature shift, transposition was achieved with a frequency of approximately 0.01% with a plasmid curing efficiency of close to 100%. Southern blot analysis demonstrated that the majority of mutants contained a single copy of Tn917 and showed no evidence for preferential sites of integration (“hot spots”). Screening of Tn917 libraries of S. pyogenes has led to the identification of mutants lacking either haemolysing or plasminogen activating activity. Mutants defective in each of these activities were identified at a frequency of approximately one in 1000 to 4000 colonies. These findings suggest that the pTV1-OK vector can be used for transposon mutagenesis of S. pyogenes and that this strategy will be valuable for identifying virulence factors and regulatory mechanisms in these bacteria.     

Genetic and physiologic analysis of a formyl-tetrahydrofolate synthetase mutant of Streptococcus mutans.

Previously we reported that transposon Tn917 mutagenesis of Streptococcus mutans JH1005 yielded an isolate detective in its normal ability to produce a mutacin (P. J. Crowley, J. D. Hillman, and A. S. Bleiweis, abstr. D55, p. 258 in Abstracts of the 95th General Meeting of the American Society for Microbiology 1995, 1995). In this report we describe the recovery of the mutated gene by shotgun cloning. Sequence analysis of insert DNA adjacent to Tn917 revealed homology to the gene encoding formyl-tetrahydrofolate synthetase (Fhs) from both prokaryotic and eukaryotic sources. In many bacteria, Fhs catalyzes the formation of 10-formyl-tetrahydrofolate, which is used directly in purine biosynthesis and formylation of Met-tRNA and indirectly in the biosynthesis of methionine, serine, glycine, and thymine. Analysis of the fhs mutant grown anaerobically in a minimal medium demonstrated that the mutant had an absolute dependency only for adenine, although addition of methionine was necessary for normal growth. Coincidently it was discovered that the mutant was sensitive to acidic pH; it grew more slowly than the parent strain on complex medium at pH 5. Complementation of the mutant with an integration vector harboring a copy of fhs restored its ability to grow in minimal medium and at acidic pH as well as to produce mutacin. This represents the first characterization of Fhs in Streptococcus.     

Use of plasmid pTV1 in transposon mutagenesis and gene cloning in Bacillus amyloliquefaciens

The plasmid pTV1, constructed in Bacillus subtilis as a tool for insertional mutagenesis by the transposon Tn917, has been transferred to Bacillus amyloliquefaciens by transduction with the phage PBS1. Insertional mutants containing Tn917 were observed in the new host. Southern blot analysis of such mutants indicated no preference for insertion sites. The copy numbers of pTV1 in B. amyloliquefaciens and B. subtilis were found to be 1.4 and 14, respectively; the plasmid is less stable against loss in B. amyloliquefaciens. The overall transposition rate in B. amyloliquefaciens is nevertheless comparable to that in B. subtilis and large numbers of mutants are readily obtained. The yield of auxotrophs was about 0.7% of all mutants, but the preponderance of glutamate auxotrophs seen in B. subtilis was not observed. A number of auxotrophs were identified as to nutritional requirements and those tested were found to be stable. Mutants deficient in extracellular proteases, amylase, and ribonuclease (barnase) were also found and the inactivated barnase gene has been cloned in Escherichia coli. It seems likely, therefore, that any B. amyloliquefaciens gene for which there is a functional test could be cloned by this technique.     

利用转座子Tn917构建单核细胞增生李斯特菌菌膜形成突变株

单核细胞增生李斯特菌菌膜形成相关基因和调控因子的分离和鉴定是阐明其菌膜形成分子机理的基础。利用原生质体转化这一方式,将带有转座子Tn917的质粒pTV1OK成功地转进了单核细胞增生李斯特菌。通过诱导Tn917转座,得到单核细胞增生李斯特菌Tn917插入突变库,转座率为10-7。经96孔细胞培养板筛选发现,菌株LM49形成菌膜能力明显大于野生型。该菌株在细胞培养板中培养４d后形成的紫色圆环的颜色明显深于野生型。用Tn917特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到相应大小的扩增产物,证实该菌株基因组中有Tn917插入。Tn917的插入使菌株LM49的菌膜形成能力增强。     

Transposon mutagenesis in Staphylococcus epidermidis using the Enterococcus faecalis transposon Tn917

We transformed a clinical Staphylococcus epidermidis isolate with the Enterococcus faecalis transposon Tn917-carrying plasmid pTV1. Loss of plasmid replication was observed at 47 degrees C. Tn917 transposes efficiently and apparently randomly. The transposition frequency could be stimulated by erythromycin. Transposon mutagenesis in S. epidermidis provides a means for genetic study of the various virulence factors of this pathogen.     

Inhibitory activity of Paenibacillus polymyxa SCE2 against human pathogenic micro-organisms

Paenibacillus polymyxa strain SCE2 was shown to inhibit the growth of different potential human pathogenic bacterial strains and fungi in vitro. To determine the genetic characterization of this antimicrobial substance, strain SCE2 was transformed with plasmid pTV32(Ts), a delivery vector for Tn917-lac. After transposition, four mutants were shown to have lost their capability to inhibit Micrococcus sp. and Staphylococcus aureus RN450, but they continued to inhibit the growth of Corynebacterium fimi NCTC7547 and Escherichia coli HB101. Hybridization experiments using the DNA of the four mutants digested with different endonucleases and pTV32(Ts) as a probe showed that the place of insertion of Tn917-lac in the chromosome was the same in mutants 4 and 36 and in mutants 31 and 59, but different between these pairs. It is thought possible that more than one antimicrobial substance is being produced by strain SCE2.     

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.     

Isolation of Gln- mutants through transposon, Tn917, mutagenesis in Bacillus brevis.

Transposon, Tn917, carried on pTV1 plasmid has been used successfully to mutagenise Bacillus brevis. The transposon showed preference for insertion at an "aro" site. A second insertional event after elimination of the preferred site with ethidium bromide/acridine orange treatment has permitted isolation of Gln- mutants in B. brevis.     

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.     

Transformation of Lactobacillus plantarum with the plasmid pTV1 by electroporation

Abstract Lactobacillus plantarum ATCC 8014 was transformed with pTV1 by electroporation using a modification of a procedure described for Escherichia coli . The plasmid pTV1 which contains the pE194 replicon from Staphylococcus aureus and transposon Tn917 from Streptococcus faecalis was shown to replicate as a high copy number plasmid in L. plantarum , and the two encoded antibiotic resistance traits were expressed. Tn917 transposed with a high frequency into plasmid DNA of L. plantarum as shown by restriction enzyme analysis and Southern hybridization studies. There are no previous reports on transposition in the lactobacilli. This system may prove to be an important tool in further work on the genetics of these organisms.     

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.     

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.     

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.