Journal of Bacteriology

Dussurget, Olivier; Timm, Juliano; Gomez, Manuel; Gold, Benjamin; Yu, Shengwei; Sabol, Sue Z.; Holmes, Randall K.; Jacobs, William R., Jr.; Smith, Issar

doi: N/Apmid: 10348851

Exochelin is the primary extracellular siderophore of Mycobacterium smegmatis , and the iron-regulated fxbA gene encodes a putative formyltransferase, an essential enzyme in the exochelin biosynthetic pathway (E. H. Fiss, Y. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557-569, 1994). We investigated the regulation of fxbA by the mycobacterial IdeR, a homolog of the Corynebacterium diphtheriae iron regulator DtxR (M. P. Schmitt, M. Predich, L. Doukhan, I. Smith, and R. K. Holmes, Infect. Immun. 63:4284-4289, 1995). Gel mobility shift experiments showed that IdeR binds to the fxbA regulatory region in the presence of divalent metals. DNase I footprinting assays indicated that IdeR binding protects a 28-bp region containing a palindromic sequence of the fxbA promoter that was identified in primer extension assays. fxbA regulation was measured in M. smegmatis wild-type and ideR mutant strains containing fxbA promoter- lacZ fusions. These experiments confirmed that fxbA expression is negatively regulated by iron and showed that inactivation of ideR results in iron-independent expression of fxbA . However, the levels of its expression in the ideR mutant were approximately 50% lower than those in the wild-type strain under iron limitation, indicating an undefined positive role of IdeR in the regulation of fxbA .

Ketela, Troy; Green, Robin; Bussey, Howard

doi: N/Apmid: 10348843

The MID2 gene of Saccharomyces cerevisiae encodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor. MID2 was isolated as a multicopy activator of the Skn7p transcription factor. Deletion of MID2 causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression of MID2 causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. -Factor hypersensitivity of mid2 mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, including PKC1 , RHO1 , WSC1 , and WSC2 . Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since mid2 wsc1 mutants are inviable on medium that does not contain osmotic support. A role for MID2 in the cell integrity pathway is further supported by the finding that MID2 is required for induction of Mpk1p tyrosine phosphorylation during exposure to -factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame, MTL1 , which encodes a protein with both structural and functional similarity to Mid2p.

Rudi, Knut; Jakobsen, Kjetill S.

doi: N/Apmid: 10348857

Complex Evolutionary Patterns of tRNA UAA Leu Group I Introns in Cyanobacterial Radiation Knut Rudi * and Kjetill S. Jakobsen * Division of General Genetics, Department of Biology, University of Oslo, 0315 Oslo, Norway ABSTRACT Based on the findings that plastids and cyanobacteria have similar group I introns inserted into tRNA UAA Leu genes, these introns have been suggested to be immobile and of ancient origin. In contrast, recent evidence suggests lateral transfer of cyanobacterial group I introns located in tRNA UAA Leu genes. In light of these new findings, we have readdressed the evolution and lateral transfer of tRNA UAA Leu group I introns in cyanobacteral radiation. We determined the presence of introns in 38 different strains, representing the major cyanobacterial lineages, and characterized the introns in 22 of the strains. Notably, two of these strains have two tRNA UAA Leu genes, with each of these genes interrupted by introns, while three of the strains have both interrupted and uninterrupted genes. Two evolutionary distinct clusters of tRNA genes, with the genes interrupted by introns belonging to two distinct intron clusters, were identified. We also compared 16S rDNA and intron evolution for both closely and distantly related strains. The distribution of the introns in the clustered groups, as defined from 16S rDNA analysis, indicates relatively recent gain and/or loss of the introns in some of these lineages. The comparative analysis also suggests differences in the phylogenetic trees for 16S rDNA and the tRNA UAA Leu group I introns. Taken together, our results show that the evolution of the intron is considerably more complex than previous studies found to be the case. We discuss, based on our results, evolutionary models involving lateral intron transfer and models involving differential loss of the intron.

Xavier, Karina B.; Peist, Ralf; Kossmann, Marina; Boos, Winfried; Santos, Helena

doi: N/Apmid: 10348846

Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis : Purification and Characterization of Key Enzymes Karina B. Xavier 1 , Ralf Peist 2 , Marina Kossmann 2 , Winfried Boos 2 , and Helena Santos 1 , * Instituto de Tecnologia Quı́mica e Biológica, Universidade Nova de Lisboa, 2780 Oeiras, Portugal, 1 and Department of Biology, University of Konstanz, D-78434 Konstanz, Germany 2 ABSTRACT Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis . Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a p -nitrophenyl-α- d -glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis , but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A K m of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.

Yamagata, Akira; Kato, Junichi; Hirota, Ryuichi; Kuroda, Akio; Ikeda, Tsukasa; Takiguchi, Noboru; Ohtake, Hisao

doi: N/Apmid: 10348848

Two plasmids were discovered in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11, which was isolated from activated sludge. The plasmids, designated pAYS and pAYL, were relatively small, being approximately 1.9 kb long. They were cryptic plasmids, having no detectable plasmid-linked antibiotic resistance or heavy metal resistance markers. The complete nucleotide sequences of pAYS and pAYL were determined, and their physical maps were constructed. There existed two major open reading frames, ORF1 in pAYS and ORF2 in pAYL, each of which was more than 500 bp long. The predicted product of ORF2 was 28% identical to part of the replication protein of a Bacillus plasmid, pBAA1. However, no significant similarity to any known protein sequences was detected with the predicted product of ORF1. pAYS and pAYL had a highly homologous region, designated HHR, of 262 bp. The overall identity was 98% between the two nucleotide sequences. Interestingly, HHR-homologous sequences were also detected in the genomes of ENI-11 and the plasmidless strain Nitrosomonas europaea IFO14298. Deletion analysis of pAYS and pAYL indicated that HHR, together with either ORF1 or ORF2, was essential for plasmid maintenance in ENI-11. To our knowledge, pAYS and pAYL are the first plasmids found in the ammonia-oxidizing autotrophic bacteria.

Ward, Doyle V.; Newton, Austin

doi: N/Apmid: 10348842

Cell Cycle Expression and Transcriptional Regulation of DNA Topoisomerase IV Genes in Caulobacter Doyle V. Ward † and Austin Newton * Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 ABSTRACT DNA replication and differentiation are closely coupled during the Caulobacter crescentus cell cycle. We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the parE and parC genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D. Ward and A. Newton, Mol. Microbiol. 26:897–910, 1997). We have examined the cell cycle regulation of parE and parC and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis. The regulation of parE and parC expression is not strictly coordinated, however. The rate of parE transcription increases ca. 20-fold during the G 1 -to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication. Transcription from the parC promoter, by contrast, is induced only two- to threefold during this cell cycle period. Steady-state ParE levels are also regulated, increasing ca. twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected. These results suggest that topo IV activity may be regulated primarily through parE expression. The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major Caulobacter sigma factor ς 73 . We also present evidence that a conserved 8-mer sequence motif located in the spacers between the −10 and −35 elements of the parE and parC promoters is required for maximum levels of parE transcription, which raises the possibility that it may function as a positive regulatory element. The pattern of parE transcription and the parE and parC promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication.

Caguiat, Jonathan J.; Watson, Alice L.; Summers, Anne O.

doi: N/Apmid: 10348859

Cd(II)-Responsive and Constitutive Mutants Implicate a Novel Domain in MerR Jonathan J. Caguiat , Alice L. Watson , and Anne O. Summers * Department of Microbiology and the Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602-2605 ABSTRACT Expression of the Tn 21 mercury resistance ( mer ) operon is controlled by a metal-sensing repressor-activator, MerR. When present, MerR always binds to the same position on the DNA (the operator merO ), repressing transcription of the structural genes merTPCAD in the absence of Hg(II) and inducing their transcription in the presence of Hg(II). Although it has two potential binding sites, the purified MerR homodimer binds only one Hg(II) ion, employing Cys82 from one monomer and Cys117 and Cys126 from the other. When MerR binds Hg(II), it changes allosterically and also distorts the merO DNA to facilitate transcriptional initiation by ς 70 RNA polymerase. Wild-type MerR is highly specific for Hg(II) and is 100- and 1,000-fold less responsive to the chemically related group 12 metals, Cd(II) and Zn(II), respectively. We sought merR mutants that respond to Cd(II) and obtained 11 Cd(II)-responsive and 5 constitutive mutants. The Cd(II)-responsive mutants, most of which had only single-residue replacements, were also repression deficient and still Hg(II) responsive but, like the wild type, were completely unresponsive to Zn(II). None of the Cd(II)-responsive mutations occurred in the DNA binding domain or replaced any of the key Cys residues. Five Cd(II)-responsive single mutations lie in the antiparallel coiled-coil domain between Cys82 and Cys117 which constitutes the dimer interface. These mutations identify 10 new positions whose alteration significantly affect MerR’s metal responsiveness or its repressor function. They give rise to specific predictions for how MerR distinguishes group 12 metals, and they refine our model of the novel domain structure of MerR. Secondary-structure predictions suggest that certain elements of this model also apply to other MerR family regulators.

Matsuno, Kiyoshi; Blais, Tessa; Serio, Alisa W.; Conway, Tyrrell; Henkin, Tina M.; Sonenshein, Abraham L.

doi: N/Apmid: 10348849

A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene ( citZ ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.

Dussurget, Olivier; Timm, Juliano; Gomez, Manuel; Gold, Benjamin; Yu, Shengwei; Sabol, Sue Z.; Holmes, Randall K.; Jacobs, William R.; Smith, Issar

doi: N/Apmid: 10348851

Transcriptional Control of the Iron-Responsive fxbA Gene by the Mycobacterial Regulator IdeR † Olivier Dussurget 1 , 2 , Juliano Timm 1 , Manuel Gomez 1 , Benjamin Gold 1 , 3 , Shengwei Yu 4 , Sue Z. Sabol 5 , Randall K. Holmes 6 , William R. Jacobs Jr. 4 , and Issar Smith 1 , * TB Center, Public Health Research Institute, 1 and Department of Microbiology, New York University Medical Center, 3 New York, New York 10016; UFR de Biochimie, Université Paris 7, 75251 Paris Cedex 05, France 2 ; Department of Microbiology and Immunology, Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York 10461 4 ; Section of Gene Structure and Regulation, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 5 ; and Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262 6 ABSTRACT Exochelin is the primary extracellular siderophore of Mycobacterium smegmatis , and the iron-regulated fxbA gene encodes a putative formyltransferase, an essential enzyme in the exochelin biosynthetic pathway (E. H. Fiss, Y. Yu, and W. R. Jacobs, Jr., Mol. Microbiol. 14:557–569, 1994). We investigated the regulation of fxbA by the mycobacterial IdeR, a homolog of the Corynebacterium diphtheriae iron regulator DtxR (M. P. Schmitt, M. Predich, L. Doukhan, I. Smith, and R. K. Holmes, Infect. Immun. 63:4284–4289, 1995). Gel mobility shift experiments showed that IdeR binds to the fxbA regulatory region in the presence of divalent metals. DNase I footprinting assays indicated that IdeR binding protects a 28-bp region containing a palindromic sequence of the fxbA promoter that was identified in primer extension assays. fxbA regulation was measured in M. smegmatis wild-type and ideR mutant strains containing fxbA promoter- lacZ fusions. These experiments confirmed that fxbA expression is negatively regulated by iron and showed that inactivation of ideR results in iron-independent expression of fxbA . However, the levels of its expression in the ideR mutant were approximately 50% lower than those in the wild-type strain under iron limitation, indicating an undefined positive role of IdeR in the regulation of fxbA .

Franklund, Clifton V.; Goldberg, Joanna B.

doi: N/Apmid: 10348873

The glutamyl-tRNA synthetase ( gltX ) gene from Pseudomonas aeruginosa was identified. A plasmid containing a 2.3-kb insert complemented the temperature-sensitive gltX mutation of Escherichia coli JP1449, and GltX activity was demonstrated. The inferred amino acid sequence of this gene showed 50.6% identity with GltX from Rhizobium meliloti .