Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence.

Pseudomonas solanacearum undergoes a spontaneous mutation that pleiotropically reduces extracellular polysaccharide (EPS) production, endoglucanase activity, and virulence and increases motility. We refer to the process that coordinately affects these traits as phenotype conversion (PC) and the resulting mutants as PC types. Previous research with the wild-type strain AW1 suggested that inactivation of a single locus could mimic phenotype conversion (T. P. Denny, F. W. Makini, and S. M. Brumbley, Mol. Plant-Microbe Interact. 1:215-223, 1988). Additional Tn5 mutagenesis of AW1 generated three more mutants (AW1-81, AW1-82, and AW1-84) that were indistinguishable from the PC type and one slightly leaky mutant (AW1-87); all four had single insertions in the same 4.0-kilobase (kb) EcoRI fragment that were responsible for the PC-like phenotype. Another insertion mutant, AW1-83, which lacks an insertion in this 4.0-kb fragment, resembled the PC type except that it was reversibly induced to produce wild-type levels of EPS when cultured adjacent to AW1. The wild-type region containing the gene that controls traits affected by phenotype conversion in AW1, designated phcA, was cloned on a 2.2-kb DNA fragment that restored all the phcA::Tn5 mutants and 11 independent spontaneous PC-type derivatives of AW1 to wild-type status. Homology with the phcA region was found in diverse wild-type strains of P. solanacearum, although restriction fragment length polymorphisms were seen. No major DNA alterations were observed in the phcA homologous region of PC types from strain AW1 or 82N. PC types from 7 of 11 conjugal strains of P. solanacearum were restored to EPS+ by phcA from AW1; however, only some PC types of strain K60 were restored, whereas others were not. We believe that a functional phcA gene is required to maintain the wild-type phenotype in P. solanacearum, and for most strains phenotype conversion results from a loss of phcA gene expression or the function of its gene product.     

The genes for the Golgi apparatus N-acetylglucosaminyltransferase and the UDP-N-acetylglucosamine transporter are contiguous in Kluyveromyces lactis

The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha(1-->2)-linked N-acetylglucosamine. Previously, Smith et al. (Smith, W. L. Nakajima, T., and Ballou, C. E. (1975) J. Biol. Chem. 250, 3426-3435) characterized two mutants, mnn2-1 and mnn2-2, which lacked terminal N-acetylglucosamine in their mannoproteins. The former mutant lacks the Golgi N-acetylglucosaminyltransferase activity, whereas the latter one was recently found to be deficient in the Golgi UDP-GlcNAc transporter activity. Analysis of extensive crossings between the two mutants led Ballou and co-workers (reference cited above) to conclude that these genes were allelic or tightly linked. We have now cloned the gene encoding the K. lactis Golgi membrane N-acetylglucosaminyltransferase by complementation of the mnn2-1 mutation and named it GNT1. The mnn2-1 mutant was transformed with a 9.5-kilobase (kb) genomic fragment previously shown to contain the gene encoding the UDP-GlcNAc transporter; transformants were isolated, and phenotypic correction was monitored after cell surface labeling with fluorescein isothiocyanate-conjugated Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescence-activated cell sorter. The above 9.5-kb DNA fragment restored the wild-type lectin binding phenotype of the transferase mutant; further subcloning of this fragment yielded a smaller one containing an opening reading frame of 1,383 bases encoding a protein of 460 amino acids with an estimated molecular mass of 53 kDa, which also restored the wild-type phenotype. Transformants had also regained the ability to transfer N-acetylglucosamine to 3-0-alpha-D-mannopyranosyl-D-mannopyranoside. The gene encoding the above transferase was found to be approximately 1 kb upstream from the previously characterized MNN2 gene encoding the UDP-GlcNAc Golgi transporter. Each gene can be transcribed independently by their own promoter. To our knowledge this is the first demonstration of two Golgi apparatus functionally related genes being contiguous in a genome.     

Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis.

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.     

Identification of the sporulation gene spoOA product of Bacillus subtilis

A 2.4-kilobase fragment of the Bacillus subtilis chromosome containing the wild-type spoOA gene derived from the phi 105dspoOA+-Bc-1 transducing phage was cloned onto plasmid pBR322 in Escherichia coli. A recombinant plasmid harboring the mutant spoOA12 allele on the 2.4-kilobase insert was also constructed from the phi 105dspoOA12-1 phage DNA and pBR322. Protein products synthesized in response to plasmid DNA in a DNA-directed cell-free system derived from E. coli were analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A protein of approximately 27,500 daltons synthesized with the recombinant plasmid DNA harboring the wild-type spoOA gene as template was not formed with the recombinant plasmid DNA harboring the spoOA12 allele. Since the spoOA12 mutation is a nonsense mutation, we conclude that the 27.5-kilodalton protein is the product of the spoOA gene.     

Cloning of rfaG, B, I, and J genes for glycosyltransferase enzymes for synthesis of the lipopolysaccharide core of Salmonella typhimurium.

R-prime plasmids carrying the pyrE-rfa-cysE region of the chromosome of Salmonella typhimurium were isolated by using the vector pULB113 (RP4::mini-Mu). One of the R-prime plasmids was used as a source of DNA to clone the rfa genes for lipopolysaccharide synthesis to pBR322. The following three hybrid plasmids were constructed: pKZ15, with a 4.0-kilobase EcoRI fragment of S. typhimurium DNA, containing the rfaG gene; pKZ27, a 9-kilobase BglII fragment with the rfaG, rfaB, and rfaI genes; and pKZ26, a 7.7-kilobase HindIII fragment with the rfaG, rfaB, rfaI, and rfaJ genes. We propose that these cloned genes code for four glycosyltransferases used for synthesis of the lipopolysaccharide core region (rfaG for glucosyltransferase I; rfaI for galactosyltransferase I; rfaB for galactosyltransferase II; and rfaJ for glucosyltransferase II). For all four genes, mutants which lacked the appropriate enzyme activity were complemented by the plasmids to give completed core lipopolysaccharide with O (somatic) side chains; for rfaG, rfaB, and rfaI, mutants gave restored or even amplified levels of the appropriate glycosyltransferase in in vitro assays. We show that the order of genes in the region is pyrE-rfaG-(rfaB-rfaI)-rfaJ-rfaL-rfaF -cysE.     

Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity

Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild-type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes.     

Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

The previous demonstration that a phosphonoacetate (PAA)-resistant (PAAr) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAAs) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAAr mutant virus. Formation of PAAr recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAAr phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. When the latter was digested with ClaI, marker rescue was not detected, suggesting that the PAAr mutation mapped near a ClaI site. The sensitive ClaI site was identified by cloning partial ClaI-EcoRI fragments and testing them in the marker rescue assay. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAAr vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.     

Cloning of the GSH1 and GSH2 genes complementing the defective biosynthesis of glutathione in the methylotrophic yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the gamma-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh- phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24 with the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme gamma-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. Both enzymes have very similar biochemical properties. To determine whether both enzymes are required for growth on chondroitin sulfate, we constructed a Bacteroides suicide vector, pE3-1, and used it to create an insertional mutation that interrupts the chondroitin lyase II gene of Bacteroides thetaiotaomicron. pE3-1 contains a 4.4-kilobase cryptic B. eggerthii plasmid (pB8-51), the Escherichia coli cloning vector pBR328, and the EcoRI D fragment from the conjugative B. fragilis plasmid pBF4. A 0.8-kilobase fragment from the center of the B. thetaiotaomicron chondroitin lyase II gene was inserted in pE3-1 to create pEG817. Although, pEG817 is stably maintained in E. coli and can be mobilized into B. thetaiotaomicron by the IncP plasmid R751, pEG817 is not maintained as a plasmid in Bacteroides spp. When pEG817 was mobilized into B. thetaiotaomicron, with selection for a drug marker on pEG817, transconjugants were obtained which had pEG817 inserted into the chondroitin lyase II gene. Western blot analysis was used to confirm that intact chondroitin lyase II is not produced in the mutant. The mutant was able to utilize chondroitin sulfate as a sole source of carbon, although no active chondroitin lyase II was produced. Thus chondroitin lyase I alone appears to be sufficient for growth on chondroitin sulfate. The mutant also had some minor changes in its outer membrane protein profile. However, there was no evidence that any of the major chondroitin sulfate-associated polypeptides in the outer membrane were affected by the insertion in the chondroitin lyase II gene.     

A sequence-specific DNA-binding factor (VF1) from Anabaena sp. strain PCC 7120 vegetative cells binds to three adjacent sites in the xisA upstream region.

A DNA-binding factor (VF1) partially purified from Anabaena sp. strain PCC 7120 vegetative cell extracts by heparin-Sepharose chromatography was found to have affinity for the xisA upstream region. The xisA gene is required for excision of an 11-kilobase element from the nifD gene during heterocyst differentiation. Previous studies of the xisA upstream sequences demonstrated that deletion of this region is required for the expression of xisA from heterologous promoters in vegetative cells. Mobility shift assays with a labeled 250-base-pair fragment containing the binding sites revealed three distinct DNA-protein complexes. Competition experiments showed that VF1 also bound to the upstream sequences of the rbcL and glnA genes, but the rbcL and glnA fragments showed only single complexes in mobility shift assays. The upstream region of the nifH gene formed a weak complex with VF1. DNase footprinting and deletion analysis of the xisA binding site mapped the binding to a 66-base-pair region containing three repeats of the consensus recognition sequence ACATT.     

Mutations that affect production of branched RNA-linked msDNA in Myxococcus xanthus.

A deletion mutation of the gene (msd-msr) for the branched RNA-linked msDNA of Myxococcus xanthus was constructed by replacing the chromosomal 0.7-kilobase (kb) SmaI-XhoI fragment encompassing msd-msr with a 1.4-kb fragment carrying a gene for kanamycin resistance. It was found that this deletion strain (delta msSX) could not produce msDNA, although it still contained another species of msDNA, mrDNA (msDNA, reduced size). No apparent differences between delta msSX and the wild-type strain were observed in terms of cell growth, morphogenesis, fruiting-body formation, or motility. Both a deletion mutation at the region 100 base pairs upstream of msd and an insertion mutation at a site 500 base pairs upstream of msd showed a significant reduction of msDNA production, indicating that there is a cis- or trans-acting positive element in this region. When the 3.5-kb BamHI fragment carrying msd-msr from Stigmatella aurantiaca was inserted into the M. xanthus chromosome, the S. aurantiaca msDNA was found to be produced in M. xanthus.     

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.     

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor.

Bacillus licheniformis penicillinase genes, penP and penI, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penI(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48 degrees C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB60E24), the coding region was concluded to be the penI gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penI promoter. The regulation of penP and penI by the repressor is discussed.