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PubMed1981

Vibrio cholerae conjugative plasmid pSJ15 contains transposable prophage dVcA1.

Evidence is presented that defective prophage dVcA1 in Vibrio cholerae strain 162 was transposed to the hybrid P::Tn1 plasmid pSJ5. Properties of the resulting conjugative plasmid, pSJ15, indicated that bacteriophage VcA1, like coliphage Mu, can insert at many sites. By analogy with other Hfr-like donors, the high-frequency, polarized chromosomal transfer mediated by plasmid pSJ15 in strain 162 appeared to depend on plasmid integration through the homologous dVcA1 sequences in both replicons. When strain 162(pSJ15) donors were mated to the nonlysogenic El Tor strain RJ1, many potential ampicillin-resistant transconjugants were zygotically induced. However, surviving transconjugants (i) were immune to phage VcA1, (ii) cotransferred immunity and ampicillin resistance to nonlysogenic recipients, and (iii) did not preferentially transfer any chromosomal markers. Recombinant plasmids that transferred wild-type VcA1 prophages were readily isolated from strain RJ1 (VcA1+) lysogens that contained plasmid pSJ15. Physical measurements revealed that plasmid pSJ15 and the recombinant plasmids were about one VcA1 genome (22 to 24 megadaltons) larger than the 51-megadalton pSJ5 plasmid. Similar Hfr-like donors were constructed by introducing plasmid pSJ15 into different strain RJ1 (VcA1+) lysogens. Transfer properties of these donors indicated that the VcA1 prophage was integrated at several sites in the strain RJ1 chromosome.

Bacteriophages↗
PubMed1985

Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli.

A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start from at least two different treponemal promoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coli, we concluded that a similar proteolytic processing takes place in both E. coli and T. pallidum. Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent than tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front of the structural genes. The nucleotide sequence of the T. pallidum tmpA gene was established. This is the first T. pallidum gene sequenced. Codon usage and the nature of transcriptional and translational signals are discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a signal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having beta-galactosidase enzyme activity as well as TmpA epitopes. The enzyme-linked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage lambda were used. In this case the protein produced was a sandwich protein consisting of 21 amino acids of the lambda cro protein, 204 amino acids of the T. pallidum TmpA protein, and 1,020 amino acids of the E. coli lambda-galactosidase. The potential use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed.

Amino Acid Sequence↗
PubMed1985

Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5.

Highly efficient promoters of coliphage T5 were identified by selecting for functional properties. Eleven such promoters belonging to all three expression classes of the phage were analyzed. Their average AT content was 75% and reached 83% in subregions of the sequences. Besides the well-known conserved sequences around -10 and -33, they exhibited homologies outside the region commonly considered to be essential for promoter function. Interestingly, the consensus hexamers around -10 (TAT AAT) and -35 (TTG ACA) were never found simultaneously within the sequence of highly efficient promoters. Several of these promoters compete extremely well for Escherichia coli RNA polymerase and can be used for the efficient in vitro synthesis of defined RNA species. In addition, some of these promoters accept 7-mGpppA as the starting dinucleotide, thus producing capped mRNA in vitro which can be utilized in various eucaryotic translation systems.

Base Sequence↗
PubMed1985

F'-coded, temperature-sensitive lambda cI857 repressor gene for easy construction and regulation of lambda promoter-dependent expression systems.

We describe the construction and properties of an F' factor which carries the temperature-sensitive cI857 allele of the repressor gene of coliphage lambda and which lacks the lambda cro function. This episome can easily be transferred to any F- and F' Escherichia coli strain, thus facilitating the construction and regulation of lambda promoter-dependent expression systems without the use of defective prophages.

Bacteriophage lambda↗
PubMed1986

Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase.

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a delta trp1 yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ung1. Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.

Cell Nucleus↗
PubMed1987

Involvement of heat shock proteins in bacteriophage Mu development.

Growth of bacteriophage Mu was severely inhibited at elevated temperature in mutants defective in the heat shock genes dnaK, groEL, and groES and in the rpoH (htpR) regulatory mutant, but not in mutants defective in the heat shock genes dnaJ or grpE; growth of a mutant of Mu deficient in functions encoded in the accessory region of the Mu genome was inhibited in the latter two host mutants. Phage production in the dnaJ mutant was restored by growth in low-salt medium. The stage in Mu development primarily affected in all except the groE mutants was phage late transcription. In contrast, the groE mutants did not support growth of Mu at any temperature; neither Mu DNA replication nor transcription was inhibited in these strains, suggesting that groE is required for phage morphogenesis as observed with several other coliphages.

Bacteriophage mu↗
PubMed1987

Processing of Escherichia coli 16S rRNA with bacteriophage lambda leader sequences.

To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed.

Bacteriophage lambda↗
PubMed1987

Cloning and characterization of the c1 repressor of Pseudomonas aeruginosa bacteriophage D3: a functional analog of phage lambda cI protein.

We cloned the gene (c1) which encodes the repressor of vegetative function of Pseudomonas aeruginosa bacteriophage D3. The cloned gene was shown to inhibit plating of D3 and the induction of D3 lysogens by UV irradiation. The efficiency of plating and prophage induction of the heteroimmune P. aeruginosa phage F116L were not affected by the presence of the cloned c1 gene of D3. When the D3 DNA fragment containing c1 was subcloned into pBR322 and introduced into Escherichia coli, it was shown to specifically inhibit the plating of phage lambda and the induction of the lambda prophage by mitomycin C. The plating of lambda imm434 phage was not affected. Analysis in minicells indicated that these effects correspond to the presence of a plasmid-encoded protein of 36,000 molecular weight. These data suggest the possibility that coliphage lambda and the P. aeruginosa phage D3 evolved from a common ancestor. The conservation of the functional similarities of their repressors may have occurred because of the advantage to these temperate phages of capitalizing on the potential of the evolutionarily conserved RecA protein to monitor the level of damage to the host genome.

Bacteriophages↗
PubMed1988

Tn5-mediated transposition of plasmid DNA after transduction to Myxococcus xanthus.

After coliphage P1-mediated transfer of Tn5-containing plasmid DNA from Escherichia coli to Myxococcus xanthus, transductants were identified which contained plasmid sequences integrated at many sites on the bacterial chromosome. The unaltered plasmid DNA sequences in these transductants were apparently flanked by intact Tn5 or IS50 sequences. These results suggest that Tn5-mediated transposition has occurred and provide a method for integrating plasmid DNA into the M. xanthus chromosome without the requirement for homologous recombination.

Chromosomes, Bacterial↗
PubMed1988

Characterization of the promoter region of the Bacillus subtilis spoIIE operon.

Mutations that define the spoIIE locus of Bacillus subtilis block sporulation at an early stage and recently were shown to prevent the proteolytic processing of sigma E (sigma 29) into its active form, an event that is believed to control critical changes in gene expression during the second hour of development. By taking advantage of two Tn917-mediated insertional mutations in spoIIE, we have cloned DNA spanning the locus. Gene disruption experiments with subcloned fragments transferred to integrational vectors revealed that the locus consisted of a single transcription unit about 2.5 kilobase pairs in size. Transcriptional lacZ fusions were used to show that expression of this transcription unit initiated at 1.5 h after the end of log-phase growth and depended upon the products of all spo0 loci. Expression was directed by a single promoter whose position was determined by high-resolution S1 protection mapping. A deletion analysis of the promoter region was also carried out, with novel integrational vectors based on derivatives of coliphage M13. The results indicated that a region of DNA extending from 183 to 118 base pairs upstream from the start point of transcription was required for full activity of the spoIIE promoter. The presumptive RNA polymerase-binding region of the promoter exhibited striking similarity to the spoIIG promoter and featured perfect but unusually spaced -10 and -35 consensus sequences for sigma A (sigma 43)-associated RNA polymerase.

Bacillus subtilis↗
PubMed1988

Sequence of the dnaB gene of Salmonella typhimurium.

The dnaB gene of Escherichia coli encodes a helicase that operates at replication forks of the bacterium and certain of its bacteriophages to produce separated strands suitable for subsequent use by primase and DNA polymerase III. Here, we present the sequence of the dnaB gene of Salmonella typhimurium, a functionally interchangeable analog of the E. coli dnaB gene. The DnaB proteins of these two organisms, inferred from the DNA sequences, are identical in length and in 93% of amino acid residues. Extended portions of the DnaB proteins are also similar to two phage-encoded DNA replication proteins: the gene 4 helicase-primase of coliphage T7 and, as reported previously (H. Backhaus and J. B. Petri, Gene 32: 289-303, 1984), the gene 12 protein of Salmonella phage P22. In contrast, little similarity was found between DnaB and either the UvrD repair helicase or transcription termination factor Rho (an RNA-DNA helicase). These results identify S. typhimurium DnaB as a member of the DnaB family of proteins by structural, as well as functional, criteria and provide the basis for the eventual identification, by mutational studies, of residues in DnaB critical for its function.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence and expression of the gene for the site-specific integration protein from bacteriophage HP1 of Haemophilus influenzae.

The nucleotide sequence of the leftmost 2,363 base pairs of the HP1 genome, which includes the attachment site (attP) and the integration region, was determined. This sequence contained an open reading frame encoding a 337-residue polypeptide, which is a member of the integrase family of site-specific recombination proteins as judged by sequence comparison. The open reading frame was located immediately adjacent to the att site and was oriented so that initiation of translation would begin distal to the att site and end in its immediate vicinity. Expression of this DNA segment in Escherichia coli provided extracts which promoted site-specific recombination between plasmids containing cloned HP1 attP and Haemophilus influenzae attB sites. This recombination was directional, since no reaction was observed between plasmids containing attR and attL sites. The reaction was stimulated by the accessory protein integration host factor of E. coli. Evidence was also obtained that the integration host factor influenced the levels of HP1 integrase expression. The deduced amino acid sequence of HP1 integrase has remarkable similarity to that deduced for the integrase of coliphage 186.

Amino Acid Sequence↗
PubMed1990

Heat shock response in mycoplasmas, genome-limited organisms.

We have measured the effect of heat shock on three mycoplasmas (Acholeplasma laidlawii K2 and JA1 and Mycoplasma capricolum Kid) and demonstrated the induction of mycoplasma heat shock proteins under these conditions. Increased synthesis of at least 5 heat shock proteins in A. laidlawii K2, 11 heat shock proteins in A. laidlawii JA1, and 7 heat shock proteins in M. capricolum was observed by electrophoretic analysis of proteins from heat-shocked cells in sodium dodecyl sulfate-polyacrylamide gels. In all three strains, major heat shock proteins (66 to 68 and 26 to 29 kilodaltons [kDa]) were found. The 66- to 68-kDa protein cross-reacted with antibody to Escherichia coli DnaK protein, suggesting that this heat shock protein has been conserved in spite of major reductions in genetic complexity during mycoplasma evolution. A. laidlawii also contained a 60-kDa protein that cross-reacted with eubacterial GroEL protein and a 40-kDa protein that cross-reacted with E. coli RecA protein. Unlike with coliphages, the mycoplasma virus L2 progeny yield was not increased when virus was plated on heat-shocked A. laidlawii host cells. However, UV-irradiated L2 virus could be host cell reactivated by both A. laidlawii SOS repair and heat shock systems.

Acholeplasma laidlawii↗
PubMed1991

RNA polymerases from Pseudomonas aeruginosa and Pseudomonas syringae respond to Escherichia coli activator proteins.

The activities of RNA polymerases (RNAPs) from Pseudomonas aeruginosa and Pseudomonas syringae were compared with that of Escherichia coli RNAP. All three enzymes are able to initiate transcription at the trpBA promoter of P. aeruginosa and at the coliphage lambda promoters, pRM and pRE, in response to heterospecific activators (TrpI protein, repressor, and cII protein, respectively). However, both Pseudomonas polymerases have less stringent requirements for promoter recognition in the absence of activators than does E. coli RNAP.

Bacterial Proteins↗
PubMed1991

Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.

The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.

Cloning, Molecular↗
PubMed1992

The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants.

Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.

Amino Acid Sequence↗
PubMed1992

Streptococcus pneumoniae DNA polymerase I lacks 3'-to-5' exonuclease activity: localization of the 5'-to-3' exonucleolytic domain.

The Streptococcus pneumoniae polA gene was altered at various positions by deletions and insertions. The polypeptides encoded by these mutant polA genes were identified in S. pneumoniae. Three of them were enzymatically active. One was a fused protein containing the first 11 amino acid residues of gene 10 from coliphage T7 and the carboxyl-terminal two-thirds of pneumococcal DNA polymerase I; it possessed only polymerase activity. The other two enzymatically active proteins, which contained 620 and 351 amino acid residues from the amino terminus, respectively, lacked polymerase activity and showed only exonuclease activity. These two polymerase-deficient proteins and the wild-type protein were hyperproduced in Escherichia coli and purified. In contrast to the DNA polymerase I of Escherichia coli but similar to the corresponding enzyme of Thermus aquaticus, the pneumococcal enzyme appeared to lack 3'-to-5' exonuclease activity. The 5'-to-3' exonuclease domain was located in the amino-terminal region of the wild-type pneumococcal protein. This exonuclease activity excised deoxyribonucleoside 5'-monophosphate from both double- and single-stranded DNAs. It degraded oligonucleotide substrates to a decameric final product.

Bacterial Proteins↗
PubMed1993

Superinfection exclusion (sieB) genes of bacteriophages P22 and lambda.

The superinfection exclusion gene (sieB) of Salmonella phage P22 was mapped with phage deletion mutants. The DNA sequence in the region was reexamined in order to find an open reading frame consistent with the deletion mapping. Several discrepancies with the previously published sequence were discovered. The revised sequence revealed a single open reading frame of 242 codons with six likely translation initiation codons. On the basis of deletion and amber mutant phenotypes, the second of these six sites was inferred to be the translation initiation site of the sieB gene. The sieB gene encodes a polypeptide with 192 amino acid residues with a calculated molecular weight of 22,442, which is in reasonable agreement with that estimated from polyacrylamide gels. The transcription start site of sieB was identified by the use of an RNase protection assay. The sieB promoter thus identified was inactivated by a 2-base substitution in its -10 hexamer. The sieB gene of coliphage lambda was also identified. The promoter for lambda sieB was identified by homology to that of P22 sieB.

Amino Acid Sequence↗