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PubMed1988

Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes.

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin O. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin O, by immunoblotting with a specific antiserum against listeriolysin O. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin O would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin O (58,000). The protein sequence is highly homologous to those of streptolysin O and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin O gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.

Amino Acid Sequence↗
PubMed1989

Construction and characterization of a Vi-positive variant of the Salmonella typhi live oral vaccine strain Ty21a.

The viaB locus coding for the Vi antigen of Salmonella typhi Ty2 was cloned on a 40.6-kilobase fragment into the cosmid vector pHC79. The live, oral, attenuated Vi-negative S. typhi Ty21a vaccine strain was transformed with the recombinant cosmid encoding the viaB locus. Homologous recombination of the viaB locus into the chromosome of S. typhi Ty21a was induced by UV irradiation, and Vi-positive recombinants were selected in the presence of D-cycloserine. One such isolate, termed WR4103, contained no plasmids or the attendant antibiotic resistance markers and expressed the Vi antigen stably. Vi antigen extracted from WR4103 was immunologically indistinguishable from Vi antigen purified from S. typhi Ty2. The only detectable difference between Ty21a and WR4103 was in the production of Vi antigen. The mean lethal doses of Ty21a and WR4103 for mice were nearly identical. Immunization of mice with WR4103 engendered a Vi antibody response and afforded complete protection against fatal infection with virulent S. typhi Ty2. Thus, S. typhi WR4103 may serve as an improved oral vaccine for protection against typhoid fever.

Animals↗
PubMed1989

Molecular analysis of lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A HindIII gene bank of Neisseria gonorrhoeae MUG116 was constructed in the cosmid vector pHC79. A cosmid (pSY81) was isolated that was able to convert N. gonorrhoeae FA5100 to reactivity with monoclonal antibody (MAb) 2-1-L8. Several MAb-reactive transformants were isolated and characterized with respect to lipooligosaccharide (LOS) production as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their ability to react with two other LOS-specific MAbs (3F11 and O6B4), and Southern blot analysis. Escherichia coli containing the clone had altered lipopolysaccharide expression as determined by electrophoretic analysis; however, no reactivity was seen with gonococcus-specific MAbs. The introduction of pSY81 into FA5100 had a pleiomorphic effect, giving rise to transformants having the full parental phenotype or transformants lacking reactivity to a combination of LOS-specific MAbs. Southern blot analysis indicated that the LOS biosynthetic mutation in FA5100 was not due to chromosomal rearrangement or large deletions.

Blotting, Southern↗
PubMed1992

Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes.

Strains of enteroaggregative Escherichia coli (EAggEC) have been implicated in several studies as important agents of persistent diarrhea among infants in the developing world. We have previously shown that the aggregative adherence (AA) property of EAggEC is associated with the presence of a 60-MDa plasmid which confers AA when introduced into E. coli HB101. Here, we report the cloning of the AA determinant from EAggEC strain 17-2 into the 21.5-kb cosmid vector pCVD301. TnphoA mutagenesis of the AA cosmid clone pJPN31 implicated an AA region of approximately 12 kb. Transmission electron microscopy of HB101 (pJPN31) revealed the presence of bundle-forming fimbriae, which were absent in AA- TnphoA insertion mutants. The presence of these fimbriae, AA, and hemagglutination (HA) of human erythrocytes were all concurrently lost by single-insertion mutations. A 14-kDa protein was seen on polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of surface shear preparations from fimbriated clones. Twelve of nineteen volunteers fed EAggEC 17-2 developed rises in antibodies to the 14-kDa protein as determined by Western blot. We have termed the cloned bundle-forming fimbriae aggregative adherence fimbriae I (AAF/I); positivity with a previously described EAggEC probe and human erythrocyte HA appear to correlate with the presence of AAF/I.

Antibodies, Bacterial↗
PubMed1992

Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB.

Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. The urease protein is encoded by two subunit genes, ureA and ureB; however, accessory genes are necessary for enzyme activity. H. pylori urease genes were isolated from a cosmid gene bank and subcloned on a 5.8-kb Sau3A partial fragment carrying ureCDAB, corresponding to four open reading frames described by A. Labigne, V. Cussac, and P. Courcoux (J. Bacteriol. 173:1920-1931, 1991). Clones were confirmed as ureas gene sequences by polymerase chain reaction amplification. The recombinant enzyme was purified from the soluble protein of French press lysates of Escherichia coli DH5 alpha(pHP402) by chromatography on DEAE-Sepharose, Phenyl-Sepharose, Mono-Q, and Superose 6 resins. Fractions containing a catalytically inactive apoenzyme were identified by an enzyme-linked immunosorbent assay (ELISA) by using antisera to native UreA (29.5 kDa) and UreB (66 kDa). Purified recombinant urease was indistinguishable from native enzyme on a Superose 6 column and on Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels. The protein reacted specifically on Western blots (immunoblots) with anti-UreA and anti-UreB antibodies and was recognized with an intensity equal to that of the native enzyme in an ELISA using human sera. Clones containing only ureA and ureB also produced an assembled but inactive enzyme. Enzyme activity was not restored by in trans complementation with cloned urease accessory gene sequences from Proteus mirabilis or Morganella morganii. H. pylori urease genes (ureCDAB) subcloned into pACYC184 were also not complemented with any of 1,000 cosmid clones containing H. pylori chromosomal sequences. However, larger clones containing 4.5 kb of DNA downstream of ureB synthesized catalytically active urease when grown in minimal medium. These data indicate that the ureA and ureB genes encoding H. pylori urease are transcribed and translated in E. coli and that these genes alone are sufficient for the synthesis and assembly of the native size enzyme. Genes downstream of ureB, however, are necessary for production of a catalytically active urease.

Apoenzymes↗
PubMed1993

Sequence and immunological characterization of a serine-rich antigen from Mycobacterium leprae.

Sera from lepromatous leprosy patients were used to screen a Mycobacterium leprae lambda gt11 library. Three positive plaques were picked, and lysogens were constructed. Immunoblot analysis showed that all of the lysogens expressed an apparently identical beta-galactosidase fusion protein which reacted strongly with the sera. The 1.7-kbp insert from one clone was subcloned into the lacZ gene in pUR290; sequence analysis of the end fused to lacZ revealed an open reading frame with no significant homology to previously published sequences. The insert was used to screen an M. leprae cosmid library, and five clones were isolated. The insert was also found to hybridize to clones expressing the M. leprae antigen which had previously been designated class III and 25L. A 1.8-kbp HindIII fragment was subcloned from one of the cosmids and sequenced. The sequence revealed a 1,227-bp open reading frame, encoding a 408-amino-acid protein with a predicted molecular mass of 42,466 Da. The protein contains amino- and carboxy-terminal hydrophobic domains and a hydrophilic central domain; the amino-terminal domain shows some homology to a 51-kDa hypothetical antigen of Mycobacterium tuberculosis, while the hydrophilic region contains a high proportion of serine residues, and we have therefore designated the protein serine-rich antigen (Sra). Some repeated motifs are present in the protein, but their significance is unknown. Seventy-eight percent of serum samples from multibacillary leprosy patients and 68% of serum samples from paucibacillary leprosy patients recognized the fusion protein, showing that this is a major M. leprae antigen. In contrast, all serum samples from endemic controls were negative, while 26% of serum samples from tuberculosis patients were weakly positive.

Amino Acid Sequence↗
PubMed1993

Expression of the Pasteurella haemolytica leukotoxin is inhibited by a locus that encodes an ATP-binding cassette homolog.

Multicopy and single-copy chromosomal fusions between the Pasteurella haemolytica leukotoxin regulatory region and the Escherichia coli beta-galactosidase gene have been constructed. These fusions were used as reporters to identify and isolate regulators of leukotoxin expression from a P. haemolytica cosmid library. A cosmid clone, which inhibited leukotoxin expression from multicopy and single-copy protein fusions, was isolated and found to contain the complete leukotoxin gene cluster plus additional upstream sequences. The locus responsible for inhibition of expression from leukotoxin-beta-galactosidase fusions was mapped within these upstream sequences, by transposon mutagenesis with Tn5, and its DNA sequence was determined. The inhibitory activity was found to be associated with a predicted 440-amino-acid reading frame (lapA) that lies within a four-gene arginine transport locus. LapA is predicted to be the nucleotide-binding component of this transport system and shares homology with the Clp family of proteases.

ATP-Dependent Proteases↗
PubMed1994

Induction of Mycobacterium avium gene expression following phagocytosis by human macrophages.

Little is known about the bacterial factors that enable pathogenic mycobacteria to survive and multiply within the macrophages of the infected host. By preparing cDNA from Mycobacterium avium bacilli grown in human-derived macrophages and in broth culture and using subtractive hybridization to remove commonly expressed genes, a procedure was developed to identify genes of M. avium that are specifically expressed when the bacilli are growing within macrophages. Total RNA was isolated from M. avium recovered 5 days after infection of human macrophages and from bacilli grown in vitro in broth. Mycobacterial mRNAs were converted to cDNA by reverse transcription. Biotin-modified cDNAs prepared from M. avium grown in broth culture were used to subtract the housekeeping genes from the cDNAs of the macrophage-derived M. avium. After each round of subtraction, a sample of the unsubtracted cDNA was amplified, labeled, and hybridized to cosmid clones of M. avium DNA. After three rounds of subtraction, the amplified DNA hybridized to approximately 1% of the cosmid clones under stringent conditions. Although the majority of the genes that are induced in phagocytized M. avium cells are expressed in the broth-grown bacilli, one DNA fragment that was identified coded for an mRNA that is highly specific for M. avium in phagosomes. This procedure will be especially useful for identifying genes that are expressed in response to growth in specific environments from organisms with genetic systems that are not well characterized.

Adaptation, Physiological↗
PubMed1994

Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli.

A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert, and the putative hyaluronidase coding sequence was subcloned into pBluescript SK as a 3.8-kb PstI-ClaI fragment (designated pJCP802). The complete nucleotide sequence of this insert was determined. The region included an open reading frame sufficient to encode a polypeptide with an M(r) of 107,751. An active hyaluronidase with an M(r) of approximately 89,000 was purified to homogeneity from E. coli DH5 alpha(pJCP802). N-terminal amino acid sequence analysis of the purified protein suggested that translation initiation was occurring primarily at a TTG codon within the major open reading frame. However, immunoblot analysis using antiserum raised against the purified 89-kDa hyaluronidase indicated that E. coli DH5 alpha(pJCP802) also expressed the 107-kDa form of the enzyme. This antiserum labelled a 107-kDa protein in partially purified hyaluronidase preparations from S. pneumoniae. The hyaluronidase activity in this pneumococcal extract was also neutralized by the antiserum.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization.

Growth of Salmonella typhimurium in a low-oxygen environment induces the ability of these bacteria to enter mammalian cells. We have carried out a search for invasion genes that are expressed under low-oxygen conditions by using Tn5lacZY transcriptional fusions. Several noninvasive oxygen-regulated lacZY insertion strains have been identified. The invasion defect in one of these noninvasive S. typhimurium strains, BJ66, has been complemented by introduction of a cosmid (pBDJ125) from an S. typhimurium SL1344 gene bank. A 1.9-kb EcoRV DNA fragment subcloned from this cosmid, containing a single open reading frame (orgA), restores the ability of BJ66 to invade mammalian cells. Comparative searches of the GenBank and EMBL sequence data banks with the nucleotide sequence of the gene and deduced amino acid sequence of the protein reveal no significant similarities. Interestingly, hybridization of an orgA gene probe with a P22 chromosomal mapping library demonstrated that the orgA gene maps to a region on the chromosome between 57.5 and 60 min where other Salmonella invasion genes have been mapped. Other enteroinvasive bacteria (Shigella flexneri, Escherichia coli, Yersinia spp., and Listeria monocytogenes) lack sequences which cross hybridize to the probe. We have compared the virulence of S. typhimurium SL1344 and an isogenic orgA mutant in a mouse model of typhoid fever. The orgA mutant was as virulent as the wild-type strain was when inoculated intraperitoneally but is significantly reduced (> 60-fold) in its ability to cause disease by an oral route of infection.

Amino Acid Sequence↗
PubMed1995

Identification of rfbA, involved in B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O5.

Previous work from this laboratory has shown that a 26-kb insert in cosmid clone pFV100, isolated from a Pseudomonas aeruginosa gene library, contained genes that could restore serotype-specific B-band lipopolysaccharide (LPS) expression in rough mutant ge6. In this study, subclones from pFV100 were made to identify genes responsible for B-band LPS synthesis. Transformation of Escherichia coli HB101 with cosmid clone pFV100 resulted in expression of P. aeruginosa serotype O5 B-band LPS, indicating the presence of an rfb cluster in pFV100. Expression of P. aeruginosa LPS could not be achieved in E. coli HB101 transformed with any of the subclones. Complementation studies of well-characterized rough mutants of P. aeruginosa PAO1 deficient in B-band LPS biosynthesis were performed with the various subclones. Subclone pFV110, containing a 1.4-kb XbaI-HindIII insert, restored B-band LPS biosynthesis in mutant AK44 (A+B-; complete core). Probing chromosomal DNA from the 20 International Antigenic Typing Scheme serotypes with the 1.4-kb insert from pFV110 in Southern hybridizations revealed a positive reaction to restriction fragments in serotypes O2, O5, O16, O20, and O18. LPS of serotypes O2, O5, O16, and O20 were shown earlier to have a similar backbone structure in their O antigen. The insert in pFV110 was sequenced, and the deduced amino acid sequence was compared with sequences of protein databases. No significant homology could be detected with any sequences in the database. Open reading frame analysis identified one region, ORF303, which could encode a 33-kDa protein. Using E. coli maxicells for protein expression, orf303 mediated the expression of a unique polypeptide with an apparent molecular mass of 32.5 kDa. The deficiency in the synthesis of B-band LPS biosynthesis in mutant AK44 is apparently complemented by the 33-kDa protein encoded by orf303. We have designated this ORF rfbA. This investigation is the first report on cloning and sequencing of an rfb gene involved specifically in O-antigen biosynthesis in P. aeruginosa PAO1.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of the tia invasion locus from enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) shares with other diarrheal pathogens the capacity to invade epithelial cell lines originating from the human ileum or colon, although the role of invasion in ETEC pathogenesis remains undefined. Two distinct loci (tia and tib) that direct noninvasive E. coli to adhere to and invade intestinal epithelial cell lines have previously been isolated from cosmid libraries of the classical ETEC strain H10407. Here, we report the molecular characterization of the tia locus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cellular fractions of E. coli DH5alpha carrying the tia-positive cosmids and recombinant plasmid subclones revealed that this locus directs the production of a 25-kDa protein (the Tia protein) that is localized to the outer membrane. The tia locus was subcloned to a maximum of 2 kb and mutagenized with bacteriophage Mud. Synthesis of this protein was directly correlated with the ability of subclones and Mud transposon mutants to adhere to and invade epithelial cells. Sequencing of the tia locus identified a 756-bp open reading frame. All transposon insertions resulting in an invasion-negative phenotype mapped to this open reading frame. The open reading frame was amplified and directionally cloned behind the lac promoter of pHG165. This construct directed DHalpha to express a 25-kDa protein and to adhere to and invade epithelial cells. The role of the tia gene in directing epithelial adherence and invasion was further assessed by the construction of chromosomal tia deletion derivatives of the parent ETEC strain, H10407. These tia deletion strains were noninvasive and lacked the ability to adhere to human ileocecal cells. The tia gene shares limited homology with the Yersinia ail locus and significant homology with the hra1 agglutinin gene cloned from a porcine ETEC strain. Additionally, tia probes hybridized to geographically diverse ETEC strains, as well as some enteropathogenic E. coli, enteroaggregative E. coli, and Shigella sonnei strains.

Amino Acid Sequence↗
PubMed1998

Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics.

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.

Chromosome Mapping↗
PubMed2006

Phase variation and genomic architecture changes in Azospirillum.

The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V(I), which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V(I) differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V(I) genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.

Azospirillum↗
PubMed1983

The wild-type gene for glutamine synthetase restores ammonia control of nitrogen fixation to Gln- (glnA) mutants of Rhodopseudomonas capsulata.

The wild-type glnA gene, coding for glutamine synthetase, was cloned from the photosynthetic bacterium Rhodopseudomonas capsulata by using a cosmid library to complement the Gln- phenotype of an Escherichia coli glnA deletion strain. The original cosmid plasmid contained 37 kilobase pairs (kbp) of R. capsulata DNA, of which only 2 kbp was necessary for Gln complementation in E. coli. A plasmid containing this 2-kbp insert was mobilized into G29, a Gln- mutant of R. capsulata which is also unable to repress nitrogenase in ammonia-containing media (Nifc phenotype). The 2-kbp fragment restored glutamine-independent growth and ammonia repression of nitrogenase, indicating that in R. capsulata, production of the signal for nitrogen repression of nif depends on the activity of the glnA gene. Repression of nitrogenase was shown, by hybridization of RNA to cloned nif DNA, to occur at the level of transcription in the wild-type and the complemented G29 strains.

Ammonia↗
PubMed1983

Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus.

From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.

Bacillus cereus↗
PubMed1983

Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12.

An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79. From it, a clone was isolated for its ability to overproduce superoxide dismutase. The enzyme overproduced was manganese superoxide dismutase, as determined by electrophoresis and antibody precipitation. Maxicell analysis and two-dimensional O'Farrell polyacrylamide gel electrophoresis demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned. Subcloning fragments from the original cosmid located the sodA gene within a 4.8-kilobase EcoRI-BamHI fragment. This fragment was inserted into a lambda phage which was deleted for the att region and consequently could only lysogenize by recombination between the cloned bacterial DNA insertion and the bacterial chromosome. Genetic mapping of the prophage in such lysogens indicated that the chromosomal sodA locus lies near 87 min on the E. coli map.

Chromosome Mapping↗
PubMed1984

Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.

Alginates↗