Journal of Bacteriology

Baker, Stefanie H.; Jin, Songmu; Aldrich, Henry C.; Howard, Gary T.; Shively, Jessup M.

doi: N/Apmid: 9696760

Insertion Mutation of the Form I cbbL Gene Encoding Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) in Thiobacillus neapolitanus Results in Expression of Form II RuBisCO, Loss of Carboxysomes, and an Increased CO 2 Requirement for Growth Stefanie H. Baker 1 , Songmu Jin 1 , † , Henry C. Aldrich 2 , Gary T. Howard 1 , ‡ , and Jessup M. Shively 1 , * Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634, 1 and Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0116 2 ABSTRACT It has been previously established that Thiobacillus neapolitanus fixes CO 2 by using a form I ribulose bisphosphate carboxylase/oxygenase (RuBisCO), that much of the enzyme is sequestered into carboxysomes, and that the genes for the enzyme, cbbL and cbbS , are part of a putative carboxysome operon. In the present study, cbbL and cbbS were cloned and sequenced. Analysis of RNA showed that cbbL and cbbS are cotranscribed on a message approximately 2,000 nucleotides in size. The insertion of a kanamycin resistance cartridge into cbbL resulted in a premature termination of transcription; a polar mutant was generated. The mutant is able to fix CO 2 , but requires a CO 2 supplement for growth. Separation of cellular proteins from both the wild type and the mutant on sucrose gradients and subsequent analysis of the RuBisCO activity in the collected fractions showed that the mutant assimilates CO 2 by using a form II RuBisCO. This was confirmed by immunoblot analysis using antibodies raised against form I and form II RuBisCOs. The mutant does not possess carboxysomes. Smaller, empty inclusions are present, but biochemical analysis indicates that if they are carboxysome related, they are not functional, i.e., do not contain RuBisCO. Northern analysis showed that some of the shell components of the carboxysome are produced, which may explain the presence of these inclusions in the mutant.

Pos, Klaas Martinus; Dimroth, Peter; Bott, Michael

doi: N/Apmid: 9696764

The Escherichia coli Citrate Carrier CitT: a Member of a Novel Eubacterial Transporter Family Related to the 2-Oxoglutarate/Malate Translocator from Spinach Chloroplasts Klaas Martinus Pos , Peter Dimroth , and Michael Bott * Mikrobiologisches Institut, Eidgenössische Technische Hochschule Zürich, CH-8092 Zürich, Switzerland ABSTRACT Under anoxic conditions in the presence of an oxidizable cosubstrate such as glucose or glycerol, Escherichia coli converts citrate to acetate and succinate. Two enzymes are specifically required for the fermentation of the tricarboxylic acid, i.e., a citrate uptake system and citrate lyase. Here we report that the open reading frame (designated citT ) located at 13.90 min on the E. coli chromosome between rna and the citrate lyase genes encodes a citrate carrier. E. coli transformed with a plasmid expressing citT was capable of aerobic growth on citrate, which provides convincing evidence for a function of CitT as a citrate carrier. Transport studies with cell suspensions of the transformed strain indicated that CitT catalyzes a homologous exchange of citrate or a heterologous exchange against succinate, fumarate, or tartrate. Since succinate is the end product of citrate fermentation in E. coli , it is likely that CitT functions in vivo as a citrate/succinate antiporter. Analysis of the primary sequence showed that CitT (487 amino acids, 53.1 kDa) is a highly hydrophobic protein with 12 putative transmembrane helices. Sequence comparisons revealed that CitT is related to the 2-oxoglutarate/malate translocator (SODiT1 gene product) from spinach chloroplasts and five bacterial gene products, none of which has yet been functionally characterized. It is suggested that the E. coli CitT protein is a member of a novel family of eubacterial transporters involved in the transport of di- and tricarboxylic acids.

Zhu, Jinsong; Kong, Renqiu; Wolk, C. Peter

doi: N/Apmid: 9696774

In Anabaena spp., synthesis of the heterocyst envelope polysaccharide, required if the cell is to fix dinitrogen under aerobic conditions, is dependent on the gene hepA . A transcriptional start site of hepA was localized 104 bp 5' from its translational initiation codon. A 765-bp open reading frame, denoted hepC , was found farther upstream. Inactivation of hepC led to constitutive expression of hepA and prevented the synthesis of heterocyst envelope polysaccharide. However, the glycolipid layer of the heterocyst envelope was synthesized. A hepK mutation blocked both the synthesis of the heterocyst envelope polysaccharide and induction of hepA . The predicted product of hepK resembles a sensory protein-histidine kinase of a two-component regulatory system. Analysis of the region between hepC and hepA indicated that DNA sequences required for the induction of hepA upon nitrogen deprivation are present between bp 574 and 440 and between bp 340 and 169 relative to the transcriptional start site of hepA . Gel mobility shift assays provided evidence that one or more proteins bind specifically to the latter sequence. The Fox box sequence downstream from hepA appeared inessential for the induction of hepA .

Poquet, Isabelle; Ehrlich, S. Dusko; Gruss, Alexandra

doi: N/Apmid: N/A

Zúñiga, Manuel; Champomier-Verges, Marie; Zagorec, Monique; Pérez-Martínez, Gaspar

doi: N/Apmid: 9696763

Structural and Functional Analysis of the Gene Cluster Encoding the Enzymes of the Arginine Deiminase Pathway of Lactobacillus sake Manuel Zúñiga 1 , † , Marie Champomier-Verges 2 , Monique Zagorec 2 , and Gaspar Pérez-Martínez 1 , * Departamento de Biotecnologı́a, Instituto de Agroquı́mica y Tecnologı́a de Alimentos (CSIC), 46100 Burjassot, Valencia, Spain, 1 and Laboratoire de Recherches sur la Viande, INRA-Jouy, Domaine de Vilvert, 78352 Jouy en Josas, France 2 ABSTRACT Lactobacillus sake can use arginine via the arginine deiminase (ADI) pathway. We designed degenerate primers based on an alignment of known sequences of ornithine transcarbamoylase (OTC)-encoding genes in order to amplify the L. sake counterpart sequences by PCR. Screening a genomic library of L. sake in λEMBL3 allowed us to isolate a clone containing a 10-kb L. sake genomic DNA insert. Sequence analysis revealed that the genes involved in arginine catabolism were clustered and encoded ADI ( arcA ), OTC ( arcB ), carbamate kinase ( arcC ), and a putative carrier with high similarity to the arginine/ornithine antiporter of Pseudomonas aeruginosa ( arcD ). Additionally, a putative transaminase-encoding gene ( arcT ) was located in this region. The genes followed the order arcA arcB arcC arcT arcD , which differs from that found in other microorganisms. arcA , arcB , arcC , and arcD mutants were constructed, and the ADI pathway was impaired in all of them. Transcriptional studies indicated that arcA gene is subject to catabolite repression, and under the conditions used, several transcripts could be detected, suggesting the existence of different initiation sites or processing of a larger mRNA.

Tourneux, Lise; Bucurenci, Nadia; Lascu, Ioan; Sakamoto, Hiroshi; Briand, Gilbert; Gilles, Anne-Marie

doi: N/Apmid: 9696781

Substitution of an Alanine Residue for Glycine 146 in TMP Kinase from Escherichia coli Is Responsible for Bacterial Hypersensitivity to Bromodeoxyuridine Lise Tourneux 1 , Nadia Bucurenci 1 , † , Ioan Lascu 2 , Hiroshi Sakamoto 1 , Gilbert Briand 3 , and Anne-Marie Gilles 1 , * Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 75724 Paris Cedex 15, 1 Institut de Biochimie et Génétique Cellulaires, Université de Bordeaux II, 33077 Bordeaux Cedex, 2 and Laboratoire d’Application de Spectrométrie de Masse, Université de Lille II, 59045 Lille Cedex, 3 France ABSTRACT The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2′-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type tmk gene from Escherichia coli or Bacillus subtilis , but not the defective tmk gene, restored the resistance to bromodeoxyuridine of an E. coli mutant strain.

Paoli, George C.; Vichivanives, Padungsri; Tabita, F. Robert

doi: N/Apmid: 9696777

Physiological Control and Regulation of the Rhodobacter capsulatus cbb Operons George C. Paoli † , Padungsri Vichivanives , and F. Robert Tabita * Department of Microbiology and Plant Molecular Biology/Biotechnology Program, The Ohio State University, Columbus, Ohio 43210-1292 ABSTRACT The genes encoding enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus are organized in at least two operons, each preceded by a separate cbbR gene, encoding potential LysR-type transcriptional activators. As a prelude to studies of cbb gene regulation in R. capsulatus , the nucleotide sequence of a 4,537-bp region, which included cbbR II , was determined. This region contained the following open reading frames: a partial pgm gene (encoding phosphoglucomutase) and a complete qor gene (encoding NADPH:quinone oxidoreductase), followed by cbbR II , cbbF (encoding fructose 1,6-bisphosphatase), cbbP (encoding phosphoribulokinase), and part of cbbT (encoding transketolase). Physiological control of the CBB pathway and regulation of the R. capsulatus cbb genes were studied by using a combination of mutant strains and promoter fusion constructs. Characterization of mutant strains revealed that either form I or form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), encoded by the cbbLS and cbbM genes, respectively, could support photoheterotrophic and autotrophic growth. A strain with disruptions in both cbbL and cbbM could not grow autotrophically and grew photoheterotrophically only when dimethyl sulfoxide was added to the culture medium. Disruption of cbbP resulted in a strain that did not synthesize form II RubisCO and had a phenotype similar to that observed in the RubisCO-minus strain, suggesting that there is only one cbbP gene in R. capsulatus and that this gene is cotranscribed with cbbM . Analysis of RubisCO activity and synthesis in strains with disruptions in either cbbR I or cbbR II , and β-galactosidase determinations from wild-type and mutant strains containing cbb Ip - and cbb IIp - lacZ fusion constructs, indicated that the cbb I and cbb II operons of R. capsulatus are within separate CbbR regulons.

Pos, Klaas Martinus; Dimroth, Peter; Bott, Michael

doi: N/Apmid: 9696764

Under anoxic conditions in the presence of an oxidizable cosubstrate such as glucose or glycerol, Escherichia coli converts citrate to acetate and succinate. Two enzymes are specifically required for the fermentation of the tricarboxylic acid, i.e., a citrate uptake system and citrate lyase. Here we report that the open reading frame (designated citT ) located at 13.90 min on the E. coli chromosome between rna and the citrate lyase genes encodes a citrate carrier. E. coli transformed with a plasmid expressing citT was capable of aerobic growth on citrate, which provides convincing evidence for a function of CitT as a citrate carrier. Transport studies with cell suspensions of the transformed strain indicated that CitT catalyzes a homologous exchange of citrate or a heterologous exchange against succinate, fumarate, or tartrate. Since succinate is the end product of citrate fermentation in E. coli , it is likely that CitT functions in vivo as a citrate/succinate antiporter. Analysis of the primary sequence showed that CitT (487 amino acids, 53.1 kDa) is a highly hydrophobic protein with 12 putative transmembrane helices. Sequence comparisons revealed that CitT is related to the 2-oxoglutarate/malate translocator (SODiT1 gene product) from spinach chloroplasts and five bacterial gene products, none of which has yet been functionally characterized. It is suggested that the E. coli CitT protein is a member of a novel family of eubacterial transporters involved in the transport of di- and tricarboxylic acids.

Darwin, Andrew J.; Ziegelhoffer, Eva C.; Kiley, Patricia J.; Stewart, Valley

doi: N/Apmid: 9696769

Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro Andrew J. Darwin 1 , † , Eva C. Ziegelhoffer 2 , ‡ , Patricia J. Kiley 3 , and Valley Stewart 1 , * Section of Microbiology, Cornell University, Ithaca, New York 14853, 1 and Departments of Bacteriology 2 and Biomolecular Chemistry, 3 University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

Dantley, Kathi A.; Dannelly, H. Kathleen; Burdett, Vickers

doi: N/Apmid: 9696754

Binding Interaction between Tet(M) and the Ribosome: Requirements for Binding Kathi A. Dantley , H. Kathleen Dannelly † , and Vickers Burdett * Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710 ABSTRACT Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5′-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.