Journal of Bacteriology

Zeilstra-Ryalls, J H; Kaplan, S

doi: N/Apmid: 7751286

Regulation of 5-aminolevulinic acid synthesis in Rhodobacter sphaeroides 2.4.1: the genetic basis of mutant H-5 auxotrophy. J H Zeilstra-Ryalls and S Kaplan Department of Microbiology and Molecular Genetics, University of Texas Health Science Center at Houston 77225, USA. ABSTRACT Rhodobacter sphaeroides H-5 was isolated as a 5-aminolevulinic acid (ALA) auxotroph following treatment of wild-type cells with N-methyl-N-nitroso-N'-nitroguanidine (J. Lascelles and T. Altshuler, J. Bacteriol. 98:721-727, 1969). The existence in R. sphaeroides 2.4.1 of the genes hemA and hemT, each encoding the enzyme 5-aminolevulinic acid synthase (EC 2.3.1.37), raised questions as to the genetic basis for the ALA auxotrophy in mutant H-5. We therefore cloned both the hemA and hemT genes from mutant H-5. The hemA gene has been sequenced in its entirety and bears four base pair substitutions which encode three amino acid changes relative to the sequence of wild-type strain 2.4.1. Complementation analysis of an Escherichia coli ALA auxotroph has revealed that the loss of ALA synthase activity in the HemA mutant enzyme could be localized to two of the amino acid substitutions. On the other hand, the hemT gene from mutant H-5 was able to complement an E. coli mutant requiring ALA for growth. Complementation analyses were also carried out by introducing the cloned hemA or hemT gene of mutant H-5 or wild-type 2.4.1 in trans into H-5 and, in parallel, into our previously described HemA-HemT double mutant strain AT1 (E. L. Neidle and S. Kaplan, J. Bacteriol. 175:2304-2313, 1993). This analysis revealed that while the complementation pattern of mutant AT1 parallels that for the E. coli ALA auxotroph, mutant H-5 could only be complemented by the wild-type hemA gene. The ability of the hemT gene of either mutant H-5 or wild-type 2.4.1 to complement the ALA auxotrophy of mutant AT1 but not mutant H-5 was consistent with beta-galactosidase activities obtained with hemT-lacZ transcriptional fusions. We conclude that the ALA auxotrophy of mutant H-5 arises from (i) a nonfunctional HemA protein containing multiple missense substitutions and (ii) an inability of the normal hemT gene to be expressed in the mutant H-5 genetic background, i.e., an additional mutation of unknown origin is required for hemT expression. These studies bear directly on the regulation of the expression of the hemA and hemT genes of R. sphaeroides 2.4.1. CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? « Previous | Next Article » Table of Contents This Article J. Bacteriol. May 1995 vol. 177 no. 10 2760-2768 » Abstract PDF Services Email this article to a colleague Similar articles in ASM journals Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of JB Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Zeilstra-Ryalls, J. H. Articles by Kaplan, S. Search for related content PubMed PubMed citation Articles by Zeilstra-Ryalls, J. H. Articles by Kaplan, S. Related Content Load related web page information Social Bookmarking CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? current issue December 2011, volume 193, issue 24 Alert me to new issues of JB About JB Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JB RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • [email protected] Print ISSN: 0021-9193 Online ISSN: 1098-5530 Copyright © 2011 by the American Society for Microbiology. For an alternate route to JB .asm.org, visit: http://intl- JB .asm.org | More Info»

Hiratsu, K; Amemura, M; Nashimoto, H; Shinagawa, H; Makino, K

doi: N/Apmid: 7751307

K Hiratsu, M Amemura, H Nashimoto, H Shinagawa and K Makino Department of Molecular Microbiology, Osaka University, Japan. In vitro transcription analysis has shown that only RNA polymerase containing an alternative sigma subunit, sigma E, activates transcription from one of the rpoH promoters and the htrA promoter. The location of the rpoE gene encoding sigma E on the Escherichia coli chromosome has recently been established, but no rpoE mutant has yet become available for phenotypic testing. We cloned the rpoE gene from the lambda-ordered clones of the E. coli genome and confirmed that the reconstituted RNA polymerase containing the gene product (E sigma E) can transcribe htrA in vitro. We constructed an rpoE-defective strain by gene disruption using the cloned rpoE gene. We demonstrate that expression of htrA is completely dependent on the rpoE gene in vivo and that the rpoE gene is essential for bacterial growth at high temperature.

Cunliffe, H E; Merriman, T R; Lamont, I L

doi: N/Apmid: 7751284

Cloning and characterization of pvdS, a gene required for pyoverdine synthesis in Pseudomonas aeruginosa: PvdS is probably an alternative sigma factor. H E Cunliffe , T R Merriman and I L Lamont Department of Biochemistry, University of Otago, Dunedin, New Zealand. ABSTRACT Cells of Pseudomonas aeruginosa secrete a fluorescent yellow-green siderophore, pyoverdine, when grown under iron-deficient conditions. We describe here the cloning and characterization of a gene, pvdS, which is required for this process. The pvdS gene is required for expression from promoters of at least two pyoverdine synthesis genes and can cause expression from these promoters in Escherichia coli, where they are otherwise inactive. Sequencing of pvdS revealed that it is a member of a subfamily of RNA polymerase sigma factors which direct the synthesis of extracellular products by bacteria. The pvdS gene is expressed only in iron-starved bacteria, and in E. coli cells at least, expression is regulated by the Fur repressor protein. We propose that in iron-rich cells of P. aeruginosa, Fur binds to the pvdS promoter and prevents expression of the gene; under conditions of iron starvation, repression is relieved and PvdS is made, reprogramming the cells for pyoverdine synthesis. CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? « Previous | Next Article » Table of Contents This Article J. Bacteriol. May 1995 vol. 177 no. 10 2744-2750 » Abstract PDF Services Email this article to a colleague Similar articles in ASM journals Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of JB Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Cunliffe, H. E. Articles by Lamont, I. L. Search for related content PubMed PubMed citation Articles by Cunliffe, H. E. Articles by Lamont, I. L. Related Content Load related web page information Social Bookmarking CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? current issue December 2011, volume 193, issue 24 Alert me to new issues of JB About JB Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JB RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • [email protected] Print ISSN: 0021-9193 Online ISSN: 1098-5530 Copyright © 2011 by the American Society for Microbiology. For an alternate route to JB .asm.org, visit: http://intl- JB .asm.org | More Info»

Vollmer, M D; Schlömann, M

doi: N/Apmid: 7751312

Conversion of 2-chloro-cis,cis-muconate and its metabolites 2-chloro- and 5-chloromuconolactone by chloromuconate cycloisomerases of pJP4 and pAC27. M D Vollmer and M Schlömann Institut für Mikrobiologie, Universität Stuttgart, Germany. ABSTRACT 2-Chloro-cis,cis-muconate, the product of ortho-cleavage of 3-chlorocatechol, was converted by purified preparations of the pJP4- and pAC27-encoded chloromuconate cycloisomerases (EC 5.5.1.7) to trans-dienelactone (trans-4-carboxymethylenebut-2-en-4-olide). The same compound was also formed when (+)-2-chloro- and (+)-5-chloromuconolactone were substrates of these enzyme preparations. Thus, the pJP4- and pAC27-encoded chloromuconate cycloisomerases are able to catalyze chloride elimination from (+)-5-chloromuconolactone. The ability to convert (+)-2-chloromuconolactone differentiates these enzymes from other groups of cycloisomerases. CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? « Previous | Next Article » Table of Contents This Article J. Bacteriol. May 1995 vol. 177 no. 10 2938-2941 » Abstract PDF Services Email this article to a colleague Similar articles in ASM journals Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of JB Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Vollmer, M. D. Articles by Schlömann, M. Search for related content PubMed PubMed citation Articles by Vollmer, M. D. Articles by Schlömann, M. Related Content Load related web page information Social Bookmarking CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? current issue December 2011, volume 193, issue 24 Alert me to new issues of JB About JB Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JB RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • [email protected] Print ISSN: 0021-9193 Online ISSN: 1098-5530 Copyright © 2011 by the American Society for Microbiology. For an alternate route to JB .asm.org, visit: http://intl- JB .asm.org | More Info»

Sweasy, J B; Chen, M; Loeb, L A

doi: N/Apmid: 7751308

DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication. J B Sweasy , M Chen and L A Loeb Yale University School of Medicine, New Haven, Connecticut 06510, USA. ABSTRACT We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication. CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? « Previous | Next Article » Table of Contents This Article J. Bacteriol. May 1995 vol. 177 no. 10 2923-2925 » Abstract PDF Services Email this article to a colleague Similar articles in ASM journals Alert me when this article is cited Alert me if a correction is posted Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of JB Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Load citing article information Citing articles via Web of Science Citing articles via Google Scholar Google Scholar Articles by Sweasy, J. B. Articles by Loeb, L. A. Search for related content PubMed PubMed citation Articles by Sweasy, J. B. Articles by Loeb, L. A. Related Content Load related web page information Social Bookmarking CiteULike Connotea Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter What's this? current issue December 2011, volume 193, issue 24 Alert me to new issues of JB About JB Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JB RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • [email protected] Print ISSN: 0021-9193 Online ISSN: 1098-5530 Copyright © 2011 by the American Society for Microbiology. For an alternate route to JB .asm.org, visit: http://intl- JB .asm.org | More Info»

Koh, YS; Roe, JH

doi: N/Apmid: 7751275

YS Koh and JH Roe Department of Microbiology, College of Natural Sciences, Seoul National University, Korea. We have isolated promoters inducible by paraquat, a superoxide radical- generating agent, from Escherichia coli, using promoter-probing plasmid pJAC4 (Y.S. Koh and J.H. Roe, Korean J. Microbiol. 31:267-273, 1993). One promoter clone pqi-5 (pqi denotes paraquat-inducible gene) was mapped at 21.8 min on the E. coli chromosome by using the Kohara phage library. We constructed an operon fusion of the lacZ gene with the pqi- 5 promoter to monitor the expression of the gene in the single-copy state. LacZ expression was induced about 7- to 13-fold by 77 to 780 microM paraquat. Other known superoxide generators such as menadione, phenazine methosulfate, and plumbagin also induced the expression of beta-galactosidase in this fusion strain. On the other hand, no significant induction was observed with treatment with hydrogen peroxide, ethanol, and heat shock. Induction of beta-galactosidase was significantly reduced by introducing a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that pqi-5 is a member of the soxRS regulon. A DNA fragment containing the pqi-5 promoter was cloned and sequenced from the Kohara phage E2E5. We identified two pqi- 5 open reading frames (ORFs); ORF-A encodes a predicted protein of 342 amino acids, and ORF-B is truncated at the cloning site. The transcription start site from the pqi-5 promoter was determined by primer extension and S1 nuclease protection analyses. Northern (RNA) and S1 analyses indicated that there are two kinds of pqi-5 transcript; one covers ORF-A only and the other covers ORF-A and possibly also ORF- B.

Xia, W; Dowhan, W

doi: N/Apmid: 7751309

W Xia and W Dowhan Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225, USA. Escherichia coli cells are dependent on the anionic phospholipid phosphatidylglycerol for cell growth. Introduction into cells of the ability to make phosphatidylinositol did not suppress the need for naturally occurring phosphatidylglycerol. Therefore, the requirement for phosphatidylglycerol must be more than maintenance of the proper membrane surface charge.

Klasen, R; Bringer-Meyer, S; Sahm, H

doi: N/Apmid: 7751271

R Klasen, S Bringer-Meyer and H Sahm Institut fur Biotechnologie, Forschungszentrum Julich, Germany. Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneity. This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5- ketogluconate. GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits. The pH optimum of gluconate oxidation was pH 10, and apparent Km values were 20.6 mM for the substrate gluconate and 73 microM for the cosubstrate NADP. The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate. D-Glucose, D- sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L- sorbose were not reduced. Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction. The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme. The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Hopper, S; Bock, A

doi: N/Apmid: 7751289

S Hopper and A Bock Lehrstuhl fur Mikrobiologie, Universitat Munchen, Germany. The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli. The protein shares homology with the sigma 54-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus lacking the aspartate residue which is the site of phosphorylation. Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA. The presence of both formate and DNA carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis. Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA. Half-maximal saturation of FHLA with formate took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM. The stimulation of ATPase activity by formate was conferred by a decrease in the apparent Km for ATP, whereas the effect of the DNA binding site also affected the Kcat of the reaction. The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA. The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate. Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity.

Paulsen, IT; Brown, MH; Dunstan, SJ; Skurray, RA

doi: N/Apmid: 7751293

IT Paulsen, MH Brown, SJ Dunstan and RA Skurray School of Biological Sciences, University of Sydney, New South Wales, Australia. The QacC polypeptide is a member of a family of small membrane proteins which confer resistance to toxic compounds. The staphylococcal qacC gene confers resistance to toxic organic cations via proton-dependent export. The membrane topology of the QacC polypeptide was investigated by constructing and analyzing a series of qacC-phoA and qacC-lacZ fusions. From these analyses, most of the predicted features of the QacC protein were verified, although data regarding the possible orientation of the COOH region were not conclusive. The role of the sole cysteine residue, Cys-42, in QacC was studied by using the sulfhydryl reagent N-ethylmaleimide and site-directed mutagenesis. N- Ethylmaleimide was shown to inhibit qacC-mediated ethidium export. Multiple amino acid substitutions were made for Cys-42, and mutations at this location had various effects on resistance specificity. This suggests that the Cys-42 residue may be located near a region of QacC that is involved in substrate recognition. Mutagenesis of conserved residues in QacC indicated that Tyr-59 and Trp-62 also play an essential structural or functional role in QacC.