Fimbriation genes of Salmonella enteritidis.

From a cosmid clone, a 5.3-kilobase (kb) HindIII fragment of Salmonella enteritidis DNA containing the fimA gene was subcloned into bacteriophage T7 promoter vectors in both orientations. Predominantly mature fimbrin (14,000 Mr) was produced by clones containing the 5.3-kb insert, whereas the original cosmid clone predominantly accumulated a prefimbrin precursor (16,500 Mr). T7 RNA polymerase-dependent expression of the 5.3-kb insert and of a series of nested deletions revealed several membrane-localized polypeptides (80,000, 40,000, 29,000, 25,000, and 16,500 Mrs) transcribed in the same direction as fimA as well as a single polypeptide (9,000 Mr) transcribed in the opposite direction. Mini-Mu and TnphoA (Tn5 IS50L::phoA) transposon mutagenesis was used to identify a 2- to 3.5-kb region downstream of fimA that affected fimbrin production and processing. A more distant region (greater than 7 kb), revealed by Tn10 and Mu dI mutagenesis, was also required for fimbriation but did not hybridize with the 5.3-kb fragment. Yet another distant region did hybridize to the 5.3-kb fragment, suggesting the existence of other fimbriation-related genes.     

浑球红细菌谷氨酸合酶基因(glt)的克隆和图谱分析

利用转座子Tn5随机插入诱变筛选得到12株浑球红细菌(Rhodobacter sphaeroides)氨同化缺陷突变株(Asm~-)。这些突变株胞内均无GOGAT活性,同时它们均无固氮酶活性(Nif~-),并且具有氮代谢多效性缺失表型(Ntr~-)。将含有Azorhizobium sesbaniae ORS571的完整glt基因的质粒pHB10转入突变株中能互补上述表型。通过筛选携带Tn5的R-prime质粒克隆了glt::Tn5片段。Southern杂交证明所克隆glt::Tn5片段与E. coli的gltBD基因有同源性。用此片段与以pLAFR3为载体所构建的R. sphaeroides 601基因文库进行菌落原位杂交筛选到了携带glt基因的cosmid pLT27。pLT27能互补所有12株R.sphaeroides氨同化缺陷突变株。酶切分析表明在该cosmid中插人的染色体DNA片段大小约为26.5kb。以pRK415为载体亚克隆了4.0kb与10.5kh的pLT27的Hindlll酶切片段,分别命名为pLTRK271与pLTRK272。pLTRK272能互补变种GT6、GT10、GT11,pLTRK…     

Transposon insertion mutagenesis of a genetic region encoding serum resistance in an 80 kb plasmid of Salmonella dublin

Using transposon insertion mutagenesis with Tn1 or Tn5, we obtained Salmonella dublin mutant strains that showed either diminished serum resistance (five mutants) or diminished mouse lethality (two mutants). Detailed restriction cleavage analysis to determine the single sites of transposon insertion in an 80 kb plasmid (pTE800) indicated that a region for serum resistance was located within a 3.0 kb region of the SalI cleavage fragment 5 and the HindIII fragment 2, while the region for mouse lethality was within a 6.0 kb region of the SalI fragment 2 and the HindIII fragment 1. When the Tn1-containing SalI fragment 5 was reconverted, by homologous recombination, to the original SalI fragment 5 (9.6 kb), serum resistance was recovered to the same level as that of a parent strain 52401. Moreover, the change in the serum resistance correlated with changes in the neutral sugar composition of the LPS. The mutation in the plasmid in strain TE4-55 that gave diminished mouse lethality was also reversed by recombination with the cloned SalI fragment 2 (15.0 kb), with concomitant recovery of mouse lethality. These results indicate that the genetic region for serum resistance is different from that for mouse lethality, and that the gene for serum resistance is closely involved with the expression of the neutral sugar composition of the LPS of S. dublin.     

Analysis of the ruv locus of Escherichia coli K-12 and identification of the gene product.

The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415. The recombinant plasmid pPVA101 fully complements the DNA repair-deficient phenotype of ruv mutants. Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region. Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment. A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000.     

Molecular characterization of a STreptococcus mutans mutant altered in environmental stress responses.

A mutant defective in aciduricity, GS5Tn1, was constructed following mutagenesis of Streptococcus mutans GS5 with the conjugative transposon Tn916. The mutant grew poorly at acidic pH levels and was sensitive to high osmolarity and elevated temperatures. These properties resulted from a single insertion of Tn916 into the GS5 chromosome, and the DNA fragment harboring the transposon was isolated into the cosmid vector, charomid 9-20. Spontaneous excision of Tn916 from the cosmid revealed that Tn916 inserted into a 8.6-kb EcoRI fragment. On the basis of the restriction analyses of insert fragments, it was found that Tn916 inserted into a 0.9-kb EcoRI-XbaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames, ORF1 and ORF2. By using a marker rescue strategy, a 6.0-kb HindIII fragment including the target site for Tn916 insertion and the 5' end of ORF1 was isolated and sequenced. The deduced amino acid sequences of ORF1 and ORF2 showed significant homology with the diacylglycerol kinase and Era proteins, respectively, from Escherichia coli. Nucleotide sequence analysis of the Tn916 insertion junction region in the GS5Tn1 chromosome revealed that the transposon inserted near the 3' terminus of ORF1. Restoration of ORF1 to its original sequence in mutant GS5Tn1 was carried out following transformation with integration vector pVA891 containing an intact ORF1. The resultant transformant showed wild-type levels of aciduricity as well as resistance to elevated temperatures and high osmolarity. These results suggest that the S. mutans homolog of diacylglycerol kinase is important for adaptation of the organism to several environmental stress signals.     

Molecular cloning, expression, and characterization of the gene for the surface (HPI)-layer protein of Deinococcus radiodurans in Escherichia coli

The HPI protein of Deinococcus radiodurans belongs to the class of surface layer proteins which form crystalline two-dimensional arrays on bacterial cell envelopes. We have cloned and expressed the gene for this protein of Mr about 100,000 by using plasmid pUC8 in Escherichia coli. As judged by immunoreaction with monospecific antibodies, apparent Mr, and limited proteolysis, a single clone contained the gene encoding the complete polypeptide on an 8.9-kilobase (kb) insert. The insert was reduced to a 5.7-kb HindIII fragment, cloned in pUC18 in both orientations, and subjected to unilateral processive deletion with exonuclease III. The library of deletion derivatives was mapped and, in conjunction with protein immunoblotting of expressed polypeptides, was used to locate the positions of the structural gene and the Deinococcus promoter region that was responsible for expression of the HPI polypeptide. The HPI gene was confined to a stretch 2.95 kb in length.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus.

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51.

Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.     

Tn4527, a Tp Sp/Sm transposon related to Tn7 and flanked by IS1

Tn4527 was isolated from a Salmonella typhimurium strain obtained in Brazil. Its size is 19.6 kb and it carries resistance to trimethoprim, spectinomycin, and streptomycin, as in the case of Tn7 (14 kb). A restriction analysis of the transposon shows regions of similarity to Tn7 mixed with extra DNA. The 2.6-kb and 2.2-kb HindIII fragments of Tn7, which encode transposition-related proteins, show homology to Tn4527. In contrast to Tn7, Tn4527 is flanked by direct repeats, which seem to be IS1's, as they have appropriate restriction sites and hybridize both to IS1 and to internal IS1 oligonucleotides.     

Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region.

Rhodobacter capsulatus mutants unable to fix nitrogen were isolated by random transposon Tn5 mutagenesis. The Tn5 insertion sites of 30 Nif- mutants were mapped within three unlinked chromosomal regions designated A, B, and C. The majority of Tn5 insertions (21 mutants) map within nif region A, characterized by two ClaI fragments of 2.5 and 25 kilobases (kb). The 17-kb ClaI fragment of nif region B contains six nif::Tn5 insertions, and the three remaining mutations are located on a 32-kb ClaI fragment of nif region C. Hybridization experiments using all 17 Klebsiella pneumoniae nif genes individually as probes revealed homology to nifE, nifS, nifA, and nifB in nif region A. The nifHDK genes were localized in nif region B. About 2 kb away from this operon, a second copy of the DNA fragments homologous to nifA and nifB, originally found in nif region A, was identified.     

Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans

A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH. A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D. radiodurans was subcloned into the EcoRI site of pAT153. The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D. radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D. radiodurans to repair proficiency with DNA derived from the gene library. Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153. The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60. The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62. None of the uvs genes introduced into the UV-sensitive E. coli CSR603 (uvrA-) was able to complement its repair defect. The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70. It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D. radiodurans genome.     

Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region.

Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.     

Characteristics of Tn3611 and Tn3613 transposons discovered in the chromosome of the BS205 strain of Pseudomonas aeruginosa

The transposons Tn3611 (HgR, 9.0 kb) and Tn3613 (SmRCmRKmRSuRHgR, 24.0 kb) were discovered in the chromosome of Pseudomonas aeruginosa BS205. The physical and genetic maps of these transposons were constructed using restriction endonucleases EcoRI, BamHI, SalI, HindIII. Genes of the mer operon, tnpA and tnpR were localized in the Tn3611 transposon, the genes DHPS-II, aad, tnpA, tnpR being situated in the map of the Tn3613 transposon. It was established that the Tn3611 belongs to the class of Tn3-like transposons and the Tn3613 is a complex transposon flanked by inverted repeats 2.2 kb in size, the transposon comprising both Tn3611 and a migrating sequence.     

Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.     

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.     

苏云金芽孢杆菌以色列亚种130kd杀蚊蛋白基因的...

The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid was confirmed by southern hybridization using a 18-base oligonucleotide probe. The crystal protein containing the component of 130kd toxic protein was purified. The crystal protein exhibiting the mosquitocidal activity against larvae of Aedes aegypti was shown by bioassay. The purified 75Md plasmid DNA of Bti 4Q5 strain was completely digested with HindIII restriction enzyme, ligated with the vector pUC18 and transformed into the recipient cells of E. coli TG1. From Apr transformants, four clones with HindIII restriction fragment inserts highly homologous to the 18-base oligonucleotide probe were obtained by in situ hybridization and southern hybridization. The 5.2kb HindIII restriction fragment insert was obtained in clone pFH2 and clone pFH4, and 2.3kb HindIII restriction fragment insert in clone pFH1 and pFH3. For pFH2 and pFH4, the 5.2kb fragment was inserted in pUC18 in opposite orientation. It contained 130kd mosquitocidal protein gene (type I) identified by restriction enzyme map analysis. The 2.3kb HindIII fragment insert in other two clones (pFH1 and pFH3) harbored a part of the type II mosquitocidal protein gene which can be used as a probe for cloning of the type II mosquitocidal protein gene.     

Cloning and characterization of the tetracycline resistance determinant of and several promoters from within the conjugative transposon Tn919

Tn919 is a 15- to 16-kilobase (kb) tetracycline resistance conjugative transposon that was originally isolated from Streptococcus sanguis FC1. The tetracycline resistance determinant (tet) was found on a 4.2-kb HindII fragment by in vitro deletion analysis. This fragment was subcloned to a pWV01 origin capable of directing replication in Escherichia coli, Bacillus subtilis, and Streptococcus lactis, and expression was observed in all three genera. In all cases, expression was weaker when only the 4.2-kb cloned fragment rather than the full transposon was present. The resistance gene is of the streptococcal tetM class and codes for a protein of approximately 70 kilodaltons. The restriction map resembles that of the tetM gene of Tn1545 (P. Martin, P. Trieu-Cuot, and P. Courvalin, Nucleic Acids Res. 14:7047-7058, 1986), which codes for a protein of 72.5 kilodaltons. A number of transposon-derived promoter-bearing fragments were also cloned and sequenced. These closely resemble the consensus sequence of E. coli and B. subtilis promoters. Fusion experiments with a truncated lacZ gene indicate the possibility of an open reading frame for one of the promoters.