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PubMed1989

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.

Bacterial Proteins↗
PubMed1989

Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli.

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.

Amino Acid Sequence↗
PubMed1990

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.

Cloning, Molecular↗
PubMed1990

Expression of the Agrobacterium tumefaciens chvB virulence region in Azospirillum spp.

Inner membranes of Azospirillum brasilense incubated with UDP-glucose were unable to synthesize beta-(1-2) glucan and lacked the 235-kilodalton intermediate protein known to be involved in the synthesis of beta-(1-2) glucan in Agrobacterium tumefaciens and Rhizobium meliloti. Inner membranes of A. brasilense strains carrying a cosmid containing the chromosomal virulence genes chvA and chvB of Agrobacterium tumefaciens formed beta-(1-2) glucan in vitro and synthesized the 235-kilodalton intermediate protein. No DNA homology to the chvB region was found in different wild-type strains of A. brasilense, but the introduction of a cosmid containing the Agrobacterium tumefaciens chvA and chvB regions yielded strains in which DNA hybridization with the chvB region was detected, provided that the strains were grown under an antibiotic selective pressure.

Blotting, Southern↗
PubMed1990

Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi.

Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.

Base Sequence↗
PubMed1990

Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187.

We recently cloned biosynthesis genes for the O7-lipopolysaccharide (O7-LPS) side chain from the Escherichia coli K-1 strain VW187 (M. A. Valvano, and J. H. Crosa, Infect. Immun. 57:937-943, 1989). To characterize the O7-LPS region, the recombinant cosmids pJHCV31 and pJHCV32 were mutagenized by transposon mutagenesis with Tn3HoHo1, which carries a promoterless lac operon and can therefore generate lacZ transcriptional fusions with target DNA sequences. Cells containing mutated plasmids were examined for their ability to react by coagglutination with O7 antiserum. The LPS pattern profiles of the insertion mutants were also investigated by electrophoresis of cell envelope fractions, followed by silver staining and immunoblotting analysis. These experiments identified three phenotypic classes of mutants and defined a region in the cloned DNA of about 14 kilobase pairs that is essential for O7-LPS expression. Analysis of beta-galactosidase production by cells carrying plasmids with transposon insertions indicated that transcription occurs in only one direction along the O7-LPS region. In vitro transcription-translation experiments revealed that the O7-LPS region encodes at least 16 polypeptides with molecular masses ranging from 20 to 48 kilodaltons. Also, the O7-LPS region in VW187 was mutagenized by homologous recombination with subsets of the cloned O7-LPS genes subcloned into a suicide plasmid vector. O7-LPS-deficient mutants of VW187 were complemented with pJHCV31 and pJHCV32, confirming that these cosmids contain genetic information that is essential for the expression of the O7 polysaccharide.

Chromosome Mapping↗
PubMed1991

Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and consequences for nodule development.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide. Mutants that fail to synthesize this exopolysaccharide are also unable to nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp. strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The background genetics and nod genes of Rhizobium sp. strain NGR234 are retained, but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. These exo genes were replaced with genes required for the synthesis of succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic manipulation, the ability of these hybrids to synthesize exopolysaccharide was restored, but the structure was that of succinoglycan and not that of Rhizobium sp. strain NGR234. The replacement genes were contained on a cosmid which encoded the entire known R. meliloti exo gene cluster, with the exception of exoB. Cosmids containing smaller portions of this exo gene cluster did not restore exopolysaccharide production. The presence of succinoglycan was indicated by staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant containing the R. meliloti exo genes produced multimers of the succinoglycan repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer. The deletion mutant therefore appeared to lack a function that affects the multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234 background. Although these hybrid strains produced succinoglycan, they were still able to induce the development of an organized nodule structure on L. leucocephala. The resulting nodules did not fix nitrogen, but they did contain infection threads and bacteroids within plant cells. This clearly demonstrated that a heterologous acidic exopolysaccharide structure was sufficient to enable nodule development to proceed beyond the developmental barrier imposed on mutants of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic exopolysaccharide.

Carbohydrate Sequence↗
PubMed1991

Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae.

Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the beta-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked Tn5 insertions and retained their resistance to the active toxin form, tabtoxnine-beta-lactam (T beta L). The wild-type DNA corresponding to the mutated region was cloned and found to restore the Tn5 mutants to toxin production. The use of cloned DNA from the region as hybridization probes revealed that the region is highly conserved among tabtoxin-producing pathovars of P. syringae and that the region deletes at a relatively high frequency (10(-3)/CFU) in BR2. The Tox- deletion mutants also lost resistance to tabtoxinine-beta-lactam. A cosmid designated pRTBL823 restored toxin production and resistance to BR2 deletion mutants. This cosmid also converted the tabtoxin-naive P. syringae epiphyte Cit7 to toxin production and resistance, indicating that pRTBL823 contains a complete set of biosynthetic and resistance genes. Tox- derivatives of BR2 did not produce disease symptoms on bean. Clones that restored toxin production to both insertion and deletion mutants also restored the ability to cause disease. However, tabtoxin-producing Cit7 derivatives remained nonpathogenic on bean and tobacco, suggesting that tabtoxin production alone is not sufficient to cause disease.

Chromosomes, Bacterial↗
PubMed1991

Molecular cloning of genes involved with expression of A-band lipopolysaccharide, an antigenically conserved form, in Pseudomonas aeruginosa.

Most strains of Pseudomonas aeruginosa can express two chemically and immunologically distinct types of lipopolysaccharide (LPS), an antigenically conserved form called A band and the serotype-specific form called B band. To study the molecular controls regulating expression of the A-band LPS antigen, we have cloned the genes involved with A-band LPS expression. Strain AK1401, a phage-resistant mutant of PAO1 which was shown previously to produce only A-band LPS and not the O-antigen-containing B-band LPS, was mutagenized by using ethyl methanesulfonate to generate an A-band-deficient mutant called rd7513. A cosmid clone bank of P. aeruginosa PAO1 whole genomic DNA was constructed in Escherichia coli. The gene bank was mobilized en masse into strain rd7513, and detection of complementation of synthesis of A band was done by screening transconjugants in a colony immunoblot assay with the A-band-specific monoclonal antibody N1F10. One recombinant cosmid, pFV3, complemented synthesis of A-band polysaccharide in rd7513. Silver-stained polyacrylamide gel and Western immunoblot analyses of LPS extracted from the transconjugant rd7513(pFV3) showed that the A band produced had a higher molecular weight than the A band of AK1401. Analysis of the plasmid pFV3 showed that it contained a chromosomal insert of 27 kb. Two subclones of pFV3, namely, pFV35 and pFV36, containing chromosomal inserts of 5.3 and 4.2 kb, respectively, also complemented A-band expression in rd7513. The LPS banding profile of rd7513(pFV35) was similar to that of AK1401, while the LPS profile of rd7513(pFV36) more closely resembled that of rd7513(pFV3). pFV3 complemented A-band expression in five of the six P. aeruginosa O serotypes which lack A band as well as in rough strain AK44 but failed to complement A-band expression in core mutants AK1012 and AK1282, suggesting that pFV3 contains genes for A-band expression and that synthesis of a complete core region in isogenic mutant strains is required for A-band synthesis.

Cloning, Molecular↗
PubMed1991

Molecular characterization and expression analysis of the anthranilate synthase gene of Pseudomonas syringae subsp. savastanoi.

The trpE gene, which encodes the large component of the enzyme anthranilate synthase, was isolated from a Pseudomonas syringae subsp. savastanoi (P. savastanoi) cosmid library. Cosmids that complemented an Escherichia coli trpE mutation contained a gene whose product is 86% homologous at the deduced amino acid level to TrpE of Pseudomonas aeruginosa and Pseudomonas putida. Amino acid sequence comparison with other TrpE sequences revealed the existence of conserved regions between the procaryotic and eucaryotic polypeptide sequences analyzed, regions that might be of functional importance. We also report on studies on the expression pattern of this gene. We analyzed the promoter activity of a trpE::lacZ transcriptional fusion, the relative amount of trpE steady-state mRNA, and the activity of anthranilate synthase from cells grown in minimal medium with or without exogenously added tryptophan and in complete medium. We concluded that under the conditions tested, expression of the trpE gene of P. savastanoi is independent of the concentration of tryptophan in the culture medium. Implications of such an expression pattern on the virulence of this bacterium are discussed.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane.

Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane (gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected. A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene. Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment. The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences. The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗
PubMed1992

Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene.

Two regulatory mutants of Pseudomonas aeruginosa, R1 and RA, that affect transcription of the pilin gene were isolated. This was done by introducing a plasmid carrying a fusion of the pilin gene's promoter with the lacZ gene into a bank of P. aeruginosa DNA mutagenized with the transposon Tn5G. The block in pilin expression in these mutants was shown to be at the level of transcription, since these mutants did not synthesize either pilin mRNA or pilin antigen. A restriction fragment derived from the R1 mutant that contains the entire transposon plus flanking chromosomal DNA was cloned and used as a probe to screen a cosmid library of P. aeruginosa DNA. Cosmids that could complement the pilin expression defect in both R1 and RA were isolated. The gene inactivated in R1 was sequenced. This gene, designated pilR, encodes an approximately 50-kDa polypeptide which exhibits significant similarity to the NtrC family of response regulators of the two-component regulatory system. PilR contains the amino-terminal aspartic acid residues which are conserved among the response regulators, suggesting that pilin gene transcription is regulated via a phosphotransfer mechanism in which PilR is phosphorylated by an as yet unidentified protein kinase.

Amino Acid Sequence↗
PubMed1992

Cloning of the outer membrane high-affinity Fe(III)-pyochelin receptor of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces the phenolic siderophore pyochelin under iron-limiting conditions. In this study, an Fe(III)-pyochelin transport-negative (Fpt-) strain, IA613, was isolated and characterized. 55Fe(III)-pyochelin transport assays determined that no Fe(III)-pyochelin associated with the Fpt- IA613 cells while a significant amount associated with KCN-poisoned Fpt+ cells. A P. aeruginosa genomic library was constructed in the IncP cosmid pLAFR1. The genomic library was mobilized into IA613, and a recombinant cosmid, pCC41, which complemented the Fpt- phenotype of IA613, was isolated. pCC41 contained a 28-kb insert of P. aeruginosa DNA, and the Fpt(-)-complementing region was localized to a 3.6-kb BamHI-EcoRI fragment by deletion and subcloning of the insert. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IA613 revealed that it lacked a 75-kDa outer membrane protein present in Fpt+ strains. IA613 strains bearing plasmid pRML303, which carries the 3.6-kb BamHI-EcoRI fragment of pCC41, expressed the 75-kDa outer membrane protein and demonstrated a 55Fe(III)-pyochelin transport phenotype identical to that of a wild-type Fpt+ strain. Minicell analysis demonstrated that the 3.6-kb BamHI-EcoRI fragment of pCC41 encoded a protein of approximately 75 kDa. The results presented here and in a previous report (D. E. Heinrichs, L. Young, and K. Poole, Infect. Immun. 59:3680-3684, 1991) lead to the conclusion that the 75-kDa outer membrane protein is the high-affinity receptor for Fe(III)-pyochelin in P. aeruginosa.

Bacterial Outer Membrane Proteins↗
PubMed1992

A conjugation procedure for Bdellovibrio bacteriovorus and its use to identify DNA sequences that enhance the plaque-forming ability of a spontaneous host-independent mutant.

Wild-type bdellovibrios are obligate intraperiplasmic parasites of other gram-negative bacteria. However, spontaneous mutants that can be cultured in the absence of host cells occur at a frequency of 10(-6) to 10(-7). Such host-independent (H-I) mutants generally display diminished intraperiplasmic-growth capabilities and form plaques that are smaller and more turbid than those formed by wild-type strains on lawns of host cells. An analysis of the gene(s) responsible for the H-I phenotype should provide significant insight into the nature of Bdellovibrio host dependence. Toward this end, a conjugation procedure to transfer both IncQ and IncP vectors from Escherichia coli to Bdellovibrio bacteriovorus was developed. It was found that IncQ-type plasmids were capable of autonomous replication in B. bacteriovorus, while IncP derivatives were not. However, IncP plasmids could be maintained in B. bacteriovorus via homologous recombination through cloned B. bacteriovorus DNA sequences. It was also found that genomic libraries of wild-type B. bacteriovorus 109J DNA constructed in the IncP cosmid pVK100 were stably maintained in E. coli; those constructed in the IncQ cosmid pBM33 were unstable. Finally, we used the conjugation procedure and the B. bacteriovorus libraries to identify a 5.6-kb BamHI fragment of wild-type B. bacteriovorus DNA that significantly enhanced the plaque-forming ability of an H-I mutant, B. bacteriovorus BB5.

Bdellovibrio↗
PubMed1992

Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria.

Many extracellular proteins produced by Erwinia chrysanthemi require the out gene products for transport across the outer membrane. In a previous report (S. Y. He, M. Lindeberg, A. K. Chatterjee, and A. Collmer, Proc. Natl. Acad. Sci. USA 88:1079-1083, 1991) cosmid pCPP2006, sufficient for secretion of Erwinia chrysanthemi extracellular proteins by Escherichia coli, was partially sequenced, revealing four out genes sharing high homology with pulH through pulK from Klebsiella oxytoca. The nucleotide sequence of eight additional out genes reveals homology with pulC through pulG, pulL, pulM, pulO, and other genes involved in secretion by various gram-negative bacteria. Although signal sequences and hydrophobic regions are generally conserved between Pul and Out proteins, four out genes contain unique inserts, a pulN homolog is not present, and outO appears to be transcribed separately from outC through outM. The sequenced region was subcloned, and an additional 7.6-kb region upstream was identified as being required for secretion in E. coli. out gene homologs were found on Erwinia carotovora cosmid clone pAKC651 but were not detected in E. coli. The outC-through-outM operon is weakly induced by polygalacturonic acid and strongly expressed in the early stationary phase. The out and pul genes are highly similar in sequence, hydropathic properties, and overall arrangement but differ in both transcriptional organization and the nature of their induction.

Amino Acid Sequence↗
PubMed1992

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.

Amino Acid Sequence↗
PubMed1993

Detection of a nitrous oxide reductase structural gene in Rhizobium meliloti strains and its location on the nod megaplasmid of JJ1c10 and SU47.

The gene encoding a denitrification enzyme, nitrous oxide reductase (EC 1.7.99.6), in Rhizobium meliloti and other gram-negative bacteria was detected by hybridization to an internal 1.2-kb PstI fragment of the structural gene (nosZ) cloned from Pseudomonas stutzeri Zobell (W.G. Zumft, A. Viebrock-Sambale, and C. Braun, Eur. J. Biochem. 192:591-599, 1990). Homology to the probe was detected in the DNAs of two N2-fixing strains of P. stutzeri, two denitrifying Pseudomonas species, one Alcaligenes eutrophus strain, and 36 of 56 R. meliloti isolates tested. Except for two isolates of R. meliloti, all showed nitrous oxide reduction activity (Nos+). Therefore, at least part of the nosZ sequence appears to be conserved and widely distributed among denitrifiers, which include free-living and symbiotic diazotrophs. By using Agrobacterium tumefaciens transconjugants harboring different megaplasmids of R. meliloti JJ1c10 and SU47, sequence homology with the nosZ probe was unequivocally located on the nod megaplasmid. A cosmid clone of JJ1c10 in which nosZ homology was mapped on a 4.2-kb BamHI fragment was selected. This cosmid, which conferred Nos+ activity to the R. meliloti wild-type strains ATCC 9930 and Balsac (Nos- and nondenitrifying, respectively) also restored Nos+ activity in the mutants of JJ1c10 and SU47 in which the 4.2-kb BamHI segment was deleted. Therefore, this segment contains sequences essential for nos gene expression in JJ1c10 and SU47 and thus confirms that the nod megaplasmid in JJ1c10 and SU47 which carries genes essential for symbiotic dinitrogen fixation also carries genes involved in the antagonistic process of denitrification.

Blotting, Southern↗
PubMed1993

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.

Amino Acid Sequence↗