bluuebunny/crossref_metadata_2025 huggingface dataset:
The Helicobacter pylori ureC gene codes for a phosphoglucosamine mutase
Authors: De Reuse H , Labigne A , Mengin-Lecreulx D
Published: November 2016

The function of UreC, the product of a 1,335-bp-long open reading frame upstream from the urease structural genes (ureAB) of Helicobacter pylori, was investigated. We present data showing that the ureC gene product is a phosphoglucosamine mutase. D. Mengin-Lecreulx and J. van Heijenoort (J. Biol. Chem. 271:32-39, 1996) observed that UreC is similar (43% identity) to the GlmM protein of Escherichia coli. Those authors showed that GlmM is a phosphoglucosamine mutase catalyzing interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, which is subsequently transformed into UDP-N-acetylglucosamine. The latter product is one of the main cytoplasmic precursors of cell wall peptidoglycan and outer membrane lipopolysaccharides. The present paper reports that, like its E. coli homolog glmM, the H. pylori ureC gene is essential for cell growth. It was known that growth of a lethal conditional glmM mutant of E. coli at a nonpermissive temperature can be restored in the presence of the ureC gene. We showed that complete complementation of the glmM mutant can be obtained with a plasmid overproducing UreC. The peptidoglycan content and the specific phosphoglucosamine mutase activity of such a complemented strain were measured; these results demonstrated that the ureC gene product functions as a phosphoglucosamine mutase. Homologs of the UreC and GlmM proteins were identified in Haemophilus influenzae, Mycobacterium leprae, Clostridium perfringens, Synechocystis sp. strain PCC6803, and Methanococcus jannaschii. Significant conservation of the amino acid sequence of these proteins in such diverse organisms suggests a very ancient common ancestor for the genes and defines a consensus motif for the phosphoglucosamine mutase active site. We propose renaming the H. pylori ureC gene the glmM gene.
Sequences and expression of pyruvate dehydrogenase genes from Pseudomonas aeruginosa
Authors: Rae J L , Cutfield J F , Lamont I L
Published: November 2016

A mutant of Pseudomonas aeruginosa, OT2100, which appeared to be defective in the production of the fluorescent yellow-green siderophore pyoverdine had been isolated previously following transposon mutagenesis (T. R. Merriman and I. L. Lamont, Gene 126:17-23, 1993). DNA from either side of the transposon insertion site was cloned, and the sequence was determined. The mutated gene had strong identity with the dihydrolipoamide acetyltransferase (E2) components of pyruvate dehydrogenase (PDH) from other bacterial species. Enzyme assays revealed that the mutant was defective in the E2 subunit of PDH, preventing assembly of a functional complex. PDH activity in OT2100 cell extracts was restored when extract from an E1 mutant was added. On the basis of this evidence, OT2100 was identified as an aceB or E2 mutant. A second gene, aceA, which is likely to encode the E1 component of PDH, was identified upstream from aceB. Transcriptional analysis revealed that aceA and aceB are expressed as a 5-kb polycistronic transcript from a promoter upstream of aceA. An intergenic region of 146 bp was located between aceA and aceB, and a 2-kb aceB transcript that originated from a promoter in the intergenic region was identified. DNA fragments upstream of aceA and aceB were shown to have promoter activities in P. aeruginosa, although only the aceA promoter was active in Escherichia coli. It is likely that the apparent pyoverdine-deficient phenotype of mutant OT2100 is a consequence of acidification of the growth medium due to accumulation of pyruvic acid in the absence of functional PDH.
A new type of hemophore-dependent heme acquisition system of Serratia marcescens reconstituted in Escherichia coli
Authors: Ghigo J M , Létoffé S , Wandersman C
Published: November 2016

The utilization by Serratia marcescens of heme bound to hemoglobin requires HasA, an extracellular heme-binding protein. This unique heme acquisition system was studied in an Escherichia coli hemA mutant that was a heme auxotroph. We identified a 92-kDa iron-regulated S. marcescens outer membrane protein, HasR, which alone enabled the E. coli hemA mutant to grow on heme or hemoglobin as a porphyrin source. The concomitant secretion of HasA by the HasR-producing hemA mutant greatly facilitates the acquisition of heme from hemoglobin. This is the first report of a synergy between an outer membrane protein and an extracellular heme-binding protein, HasA, acting as a heme carrier, which we termed a hemophore.
Specific detection of Salmonella typhimurium proteins synthesized intracellularly
Authors: Burns-Keliher L L , Portteus A , Curtiss R
Published: November 2016

Studies of the proteins Salmonella typhimurium synthesizes under conditions designed to more closely approximate the in vivo environment, i.e., in cell and tissue culture, are not easily interpreted because they have involved chemical inhibition of host cell protein synthesis during infection. The method which we have developed allows specific labeling of bacterial proteins without interfering with host cell metabolic activities by using a labeled lysine precursor which mammalian cells cannot utilize. We have resolved the labeled proteins using two-dimensional electrophoresis and autofluorography. We were able to detect 57 proteins synthesized by S. typhimurium during growth within a human intestinal epithelial cell line. Of the 57 proteins detected, 34 appear to be unique to the intracellular environment, i.e., they are not seen during growth of the bacteria in tissue culture medium alone. Current (and future) efforts are directed at organizing the 34 proteins into known stress response groups, determining the cellular locations of the proteins (outer or inner membrane, etc.), and comparing the pattern of proteins synthesized within an intestinal epithelial cell to the pattern synthesized during growth within other tissues.
2-chloromuconate and ClcR-mediated activation of the clcABD operon: in vitro transcriptional and DNase I footprint analyses
Authors: McFall S M , Parsek M R , Chakrabarty A M
Published: November 2016

In Pseudomonas putida, the plasmid-borne clcABD operon encodes enzymes involved in 3-chlorocatechol degradation. Previous studies have demonstrated that these enzymes are induced when P. putida is grown in the presence of 3-chlorobenzoate, which is converted to 3-chlorocatechol, and that ClcR, a LysR-type regulator, is required for this induction. The clcABD operon is believed to have evolved from the chromosomal catBCA operon, which encodes enzymes that utilize catechol and is regulated by CatR. The inducer for the catBCA operon is an intermediate of the catechol pathway, cis,cis-muconate. In this study, we demonstrate by the use of in vitro transcription assays and lacZ transcription fusions in vivo that the analogous intermediate of the 3-chlorocatechol pathway, 2-chloromuconate, is the inducer of the clcABD operon. The DNase I footprints of ClcR with and without 2-chloromuconate were also determined. An extended region of the promoter from -79 to -25 was occupied in the absence of inducer, but the -35 region was unprotected. When 2-chloromuconate was added to the binding assays, the footprint contracted approximately 4 bp at the proximal end of the promoter, and the -35 region was contacted. It is interesting to note that CatR actually extends its footprint 14 bp on the catBCA promoter in response to its inducer. Although CatR and ClcR change their nucleotide protection patterns in different manners when exposed to their respective inducers, their final footprints resemble each other. Therefore, it is possible that their transcriptional activation mechanisms may be evolutionarily conserved.
Composition and primary structure of the F1F0 ATP synthase from the obligately anaerobic bacterium Clostridium thermoaceticum
Authors: Das A , Ljungdahl L G
Published: November 2016

The subunit composition and primary structure of the proton-translocating F1F0 ATP synthase have been determined in Clostridium thermoaceticum. The isolated enzyme has a subunit composition identical to that of the F1F0 ATP synthase purified from Clostridium thermoautotrophicum (A. Das, D. M. Ivey, and L. G. Ljungdahl, J. Bacteriol. 179:1714-1720, 1997), both having six different polypeptides. The molecular masses of the six subunits were 60, 50, 32, 17, 19, and 8 kDa, and they were identified as alpha, beta, gamma, delta, epsilon, and c, respectively, based on their reactivity with antibodies against the F1 ATPase purified from C. thermoautotrophicum and by comparing their N-terminal amino acid sequences with that deduced from the cloned genes of the C. thermoaceticum atp operon. The subunits a and b found in many bacterial ATP synthases could not be detected either in the purified ATP synthase or crude membranes of C. thermoaceticum. The C. thermoaceticum atp operon contained nine genes arranged in the order atpI (i), atpB (a), atpE (c), atpF (b), atpH (delta), atpA (alpha), atpG (gamma), atpD (beta), and atpC (epsilon). The deduced protein sequences of the C. thermoaceticum ATP synthase subunits were comparable with those of the corresponding subunits from Escherichia coli, thermophilic Bacillus strain PS3, Rhodospirillum rubrum, spinach chloroplasts, and the cyanobacterium Synechococcus strain PCC 6716. The analysis of total RNA by Northern hybridization experiments reveals the presence of transcripts (mRNA) of the genes i, a, and b subunits not found in the isolated enzyme. Analysis of the nucleotide sequence of the atp genes reveals overlap of the structural genes for the i and a subunits and the presence of secondary structures (in the b gene) which could influence the posttranscriptional regulation of the corresponding genes.
Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4
Authors: Ito M , Guffanti A A , Zemsky J , Ivey D M , Krulwich T A
Published: November 2016

Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.
In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus
Authors: Wallace K K , Lobo S , Han L , McArthur H A , Reynolds K A
Published: November 2016

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.
Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX
Authors: Loprasert S , Atichartpongkun S , Whangsuk W , Mongkolsuk S
Published: November 2016

From Xanthomonas campestris pv. phaseoli, we have isolated by two independent methods genes involved in peroxide detoxification (ahpC and ahpF), a gene involved in peroxide sensing and transcription regulation (oxyR), and a gene of unknown function (orfX). Amino acid sequence analysis of AhpC, AhpF, and OxyR showed high identity with bacterial homologs. OrfX was a small cysteine-rich protein with no significant homology to known proteins. The genes ahpC, ahpF, oxyR, and orfX were arranged in a head-to-tail fashion. This unique arrangement was conserved in all of the Xanthomonas strains tested. The functionalities of both the ahpC and oxyR genes were demonstrated. In X. campestris pv. phaseoli, increased expression of ahpC alone conferred partial protection against growth retardation and killing by organic hydroperoxides but not by H2O2 or superoxide generators. These genes are likely to have important physiological roles in protection against peroxide toxicity in Xanthomonas.
sar Genetic determinants necessary for transcription of RNAII and RNAIII in the agr locus of Staphylococcus aureus
Authors: Cheung A L , Bayer M G , Heinrichs J H
Published: November 2016

The temporal expression of most virulence factors in Staphylococcus aureus is regulated by pleiotropic loci such as agr and sar. We have previously shown that the sar locus affects hemolysin production because it is required for agr transcription. To delineate the sar genetic determinant required for agr transcription, single copies of fragments from the sar locus, encompassing the individual sar transcripts (sarA, sarC, and sarB), were introduced into a sar mutant via the integration vector pCL84. Although a DNA fragment encompassing the sarA transcript plus a 189-bp upstream region was sufficient for agr expression, complementation analysis revealed that the sarB transcript was the most effective in augmenting agr transcription as determined by RNAII and RNAIII transcription and gel retardation assays with the P2 and P3 promoters of agr. As the region upstream of the sarA transcript encodes a 39-amino-acid open reading frame, ORF3, it is possible that posttranslational cooperation between the sarA gene product and ORF3 may be necessary for optimal agr expression. Deletion studies demonstrated that an intact sarA gene is essential for agr transcription. However, mutagenesis and in vitro translation studies revealed that unlike the agr locus, the required element is the SarA protein and not the RNA molecule. Taken together, these results indicate that the sarA-encoded protein, possibly in conjunction with peptides encoded in the upstream region, regulates hemolysin production by controlling agr P2 and P3 transcription.
Acyl-acyl carrier protein is a donor of fatty acids in the NodA-dependent step in biosynthesis of lipochitin oligosaccharides by rhizobia
Authors: Ritsema T , Lugtenberg B J , Spaink H P
Published: November 2016

NodA controls transfer of a fatty acid in the biosynthesis of lipochitin oligosaccharides by rhizobia. In an in vitro assay, we used de-N-acetylated chitin oligosaccharides substituted with an O-acetyl moiety as acyl acceptor substrates. We show that acyl-acyl carrier protein is used as a donor in NodA-directed fatty acid transfer.
Structural studies of malate dehydrogenases (MDHs): MDHs in Brevundimonas species are the first reported MDHs in Proteobacteria which resemble lactate dehydrogenases in primary structure
Authors: Charnock C
Published: November 2016

The N-terminal sequences of malate dehydrogenases from 10 bacterial strains, representing seven genera of Proteobacteria, were determined. Of these, the enzyme sequences of species classified in the genus Brevundimonas clearly resembled those malate dehydrogenases with greatest similarity to lactate dehydrogenases. Additional evidence from subunit molecular weights, peptide mapping, and enzyme mobilities suggested that malate dehydrogenases from species of the genus Brevundimonas were structurally distinct from others in the study.
Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis
Authors: Mio T , Adachi-Shimizu M , Tachibana Y , Tabuchi H , Inoue S B , Yabe T , Yamada-Okabe T , Arisawa M , Watanabe T , Yamada-Okabe H
Published: November 2016

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase.
Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan
Authors: Payie K G , Clarke A J
Published: November 2016

The relationship between the acetylation of peptidoglycan and that of aminoglycosides in Providencia stuartii has been investigated both in vivo and in vitro. Adaptation of the assay for peptidoglycan N-->O-acetyltransferase permitted an investigation of the use of peptidoglycan as a source of acetate for the N acetylation of aminoglycosides by gentamicin N-acetyltransferase [EC 2.3.1.59; AAC(2')]. The peptidoglycan from cells of P. stuartii PR50 was prelabelled with 3H by growth in the presence of N-[acetyl-3H]glucosamine. Under these conditions, [3H]acetate was confirmed to be transferred to the C-6 position of peptidoglycan-bound N-acetylmuramyl residues. Isolated cells were subsequently incubated in the presence of various concentrations of gentamicin and tobramycin (0 to 5x MIC). Analysis of various cellular fractions from isolated cells and spent culture medium by the aminoglycoside-binding phosphocellulose paper assay revealed increasing levels of radioactivity associated with the filters used for whole-cell sonicates of cells treated with gentamicin up to 2 x MIC. Beyond this concentration, a decrease in radioactivity was observed, consistent with the onset of cell lysis. Similar results were obtained with tobramycin, but the increasing trend was less obvious. The transfer of radiolabel to either aminoglycoside was not observed with P. stuartii PR100, a strain that is devoid of AAC(2')-Ia. A high-performance anion-exchange chromatography-based method was established to further characterize the AAC(2')-Ia-catalyzed acetylation of aminoglycosides. The high-performance liquid chromatography (HPLC)-based method resolved a tobramycin preparation into two peaks, both of which were collected and confirmed by 1H nuclear magnetic resonance to be the antibiotic. Authentic standards of 2'-N-acetyltobramycin were prepared and were well separated from the parent antibiotic when subjected to the HPLC analysis. By applying this technique, the transfer of radiolabelled acetate from the cell wall polymer peptidoglycan to tobramycin was confirmed. In addition, isolated and purified AAC(2')-Ia was shown to catalyze in vitro the transfer of acetate from acetyl-coenzyme A, soluble fragments of peptidoglycan, and N-acetylglucosamine to tobramycin. These data further support the proposal that AAC(2')-Ia from P. stuartii may have a physiological role in its secondary metabolism and that its activity on aminoglycosides is simply fortuitous.
The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae
Authors: Lucas C E , Balthazar J T , Hagman K E , Shafer W M
Published: November 2016

Gonococcal resistance to antimicrobial hydrophobic agents (HAs) is due to energy-dependent removal of HAs from the bacterial cell by the MtrCDE membrane-associated efflux pump. The mtrR (multiple transferrable resistance Regulator) gene encodes a putative transcriptional repressor protein (MtrR) believed to be responsible for regulation of mtrCDE gene expression. Gel mobility shift and DNase I footprint assays that used a maltose-binding protein (MBP)-MtrR fusion protein demonstrated that the MtrR repressor is capable of specifically binding the DNA sequence between the mtrR and mtrC genes. This binding site was localized to a 26-nucleotide stretch that includes the promoter utilized for mtrCDE transcription and, on the complementary strand, a 22-nucleotide stretch that contains the -35 region of the mtrR promoter. A single transition mutation (A-->G) within the MtrR-binding site decreased the affinity of the target DNA for MtrR and enhanced gonococcal resistance to HAs when introduced into HA-susceptible strain FA19 by transformation. Since this mutation enhanced expression of the mtrCDE gene complex but decreased expression of the mtrR gene, the data are consistent with the notion that MtrR acts as a transcriptional repressor of the mtrCDE efflux pump protein genes.
Uridylylation of the P(II) protein in the photosynthetic bacterium Rhodospirillum rubrum
Authors: Johansson M , Nordlund S
Published: November 2016

The regulatory protein P(II) has been studied in great detail in enteric bacteria; however, its function in photosynthetic bacteria has not been clearly established. As a number of these bacteria have been shown to regulate nitrogenase activity by a metabolic control system, it is of special interest to establish the role of P(II) in these diazotrophs. In this study, we show that P(II) in Rhodospirillum rubrum is modified in response to the N status in the cell and that addition of ammonium or glutamine leads to demodification. We also provide evidence that P(II) is uridylylated. In addition, we show that not only these compounds but also NAD+ promotes demodification of P(II), which is of particular interest as this pyridine nucleotide has been shown to act as a switch-off effector of nitrogenase. Demodification of P(II) by ammonium or NAD+ did not occur in cultures treated with an inhibitor of glutamine synthetase (methionine sulfoximine), whereas treatment with the glutamate synthase inhibitor 6-diazo-5-oxo-norleucine led to total demodification of P(II) without any other addition. The results indicate that P(II) probably is not directly involved in darkness switch-off of nitrogenase but that a role in ammonium switch-off cannot be excluded.
Promoters and transcripts associated with the aroP gene of Escherichia coli
Authors: Wang P , Yang J , Pittard A J
Published: November 2016

Analysis of in vitro transcriptional events initiating within the region immediately upstream of the aroP coding region has revealed the presence of three promoters, P1, P2, and P3. Both P1 and P2 give rise to mRNA encoding the AroP protein, whereas P3 initiates transcription in the opposite direction. Both P1 and P3 contain UP elements which contribute to promoter strength. Regulation of expression from these three promoters has been examined in vitro by using supercoiled DNA templates and in vivo by using lacZ transcriptional fusions and specific promoter mutants. Expression from P2 is partially repressed by TyrR alone both in vitro and in vivo. Addition of the aromatic amino acid tyrosine, phenylalanine, or tryptophan further increases this repression. P1 is not repressed by TyrR alone but is repressed in vivo in the presence of phenylalanine, tyrosine, or tryptophan. This also occurs in vitro but requires Ca2+ ions in the reaction mixture for its demonstration. Under these conditions, transcription from P3 is enhanced by TyrR protein with phenylalanine, tyrosine, or tryptophan. However, we were unable to demonstrate P3 expression in vivo. Under repressing conditions, there is no production of truncated RNA molecules (from P1), which would be expected if repression involved a roadblock mechanism.
Repression of the aroP gene of Escherichia coli involves activation of a divergent promoter
Authors: Wang P , Yang J , Lawley B , Pittard A J
Published: November 2016

The repression of aroP expression which is mediated by the TyrR protein with phenylalanine, tyrosine, or tryptophan has been shown to be primarily a direct result of TyrR-mediated activation of a divergent promoter, P3, which directs the RNA polymerase away from promoter P1. Evidence which has been presented to support this conclusion is as follows. Repression of P1 does not occur either in vitro or in vivo if wild-type TyrR protein is substituted by the activation-negative mutant RQ10 (with an R-to-Q change at position 10). Repression of P1 is greatly diminished if the P3 promoter is inactivated or if a 5-bp insertion is made between the P3 promoter and the binding sites for TyrR. Repression is also abolished if the promoter strength of P1 is increased or a putative UP element associated with P3 is altered. Repression of the second promoter, P2, still occurs if the wild-type TyrR protein is substituted with RQ10 or EQ274. The tryptophan-mediated repression of aroP does not involve the TrpR protein.
Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome
Authors: Hayashi H , Takagi K I , Fukumura M , Kimura T , Karita S , Sakka K , Ohmiya K
Published: November 2016

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.
Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox
Authors: Magrini V , Salmi D , Thomas D , Herbert S K , Hartzell P L , Youderian P
Published: November 2016

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.
Characterization of cp18, a naturally truncated member of the cp32 family of Borrelia burgdorferi plasmids
Authors: Stevenson B , Casjens S , van Vugt R , Porcella S F , Tilly K , Bono J L , Rosa P
Published: November 2016

We have mapped the genes encoding the antigenic lipoproteins OspE and OspF to an approximately 18-kb circular plasmid in Borrelia burgdorferi N40. Sequencing and restriction mapping have revealed that this plasmid, cp18, is homologous to an 18-kb region of the cp32 circular plasmids found in the Lyme disease spirochetes. Our data show that cp18 may have arisen from an ancestral cp32 plasmid by deletion of a 14-kb region of DNA, indicating that a significant portion of the cp32 plasmid is not essential in cis for plasmid maintenance. These findings suggest that a relatively small recombinant plasmid capable of being stably maintained in B. burgdorferi could be constructed from a cp32 plasmid.
Aerobic regulation of isocitrate dehydrogenase gene (icd) expression in Escherichia coli by the arcA and fnr gene products
Authors: Chao G , Shen J , Tseng C P , Park S J , Gunsalus R P
Published: November 2016

Isocitrate dehydrogenase, the icd gene product, has been studied extensively regarding the regulation of enzymatic activity and its relationship to the metabolic flux between the tricarboxylic acid cycle and the glyoxylate bypass. In this study, the transcriptional regulation of icd gene expression was monitored by using an icd-lacZ gene fusion and shown to vary over a 15-fold range in response to changes in oxygen and carbon availability. Anaerobic cell growth resulted in fivefold-lower icd-lacZ expression than during aerobic growth. This negative control is mediated by the arcA and fnr gene products. When different carbon compounds were used for cell growth, icd-lacZ expression varied threefold. The results of continuous cell culture studies indicated that this control may be due to variations in cell growth rate rather than to catabolite repression. DNase I footprinting at the icd promoter revealed a 42-bp ArcA-phosphate-protected region that overlaps the start site of icd transcription. Phosphorylation of ArcA considerably enhanced its binding to DNA, while ArcA-phosphate exhibited an apparent dissociation value of approximately 0.1 microM. Based on these studies, ArcA appears to function as a classical repressor of transcription by binding at a site overlapping the icd promoter during anaerobic cell growth conditions.
A melibiose transporter and an operon containing its gene in Enterobacter cloacae
Authors: Okazaki N , Jue X X , Miyake H , Kuroda M , Shimamoto T , Tsuchiya T
Published: November 2016

We detected inducible melibiose transport activity in cells of Enterobacter cloacae IID977. H+, but not Na+, was found to be the coupling cation for this transporter. We cloned and sequenced the gene encoding the melibiose transporter. A homology search of a protein sequence database revealed that this melibiose transporter has high sequence similarity with the lactose transporter (LacY) and the raffinose transporter (RafB) and has some similarity with the melibiose transporter (MelB) of Escherichia coli.
Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition
Authors: Duwat P , Cochu A , Ehrlich S D , Gruss A
Published: November 2016

Studies of cellular responses to DNA-damaging agents, mostly in Escherichia coli, have revealed numerous genes and pathways involved in DNA repair. However, other species, particularly those which exist under different environmental conditions than does E. coli, may have rather different responses. Here, we identify and characterize genes involved in DNA repair in a gram-positive plant and dairy bacterium, Lactococcus lactis. Lactococcal strain MG1363 was mutagenized with transposition vector pG+host9::ISS1, and 18 mutants sensitive to mitomycin and UV were isolated at 37 degrees C. DNA sequence analyses allowed the identification of 11 loci and showed that insertions are within genes implicated in DNA metabolism (polA, hexB, and deoB), cell envelope formation (gerC and dltD), various metabolic pathways (arcD, bglA, gidA, hgrP, metB, and proA), and, for seven mutants, nonidentified open reading frames. Seven mutants were chosen for further characterization. They were shown to be UV sensitive at 30 degrees C (the optimal growth temperature of L. lactis); three (gidA, polA, and uvs-75) were affected in their capacity to mediate homologous recombination. Our results indicate that UV resistance of the lactococcal strain can be attributed in part to DNA repair but also suggest that other factors, such as cell envelope composition, may be important in mediating resistance to mutagenic stress.
Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline
Authors: Sakai Y , Murdanoto A P , Konishi T , Iwamatsu A , Kato N
Published: November 2016

The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.
The kdp system of Clostridium acetobutylicum: cloning, sequencing, and transcriptional regulation in response to potassium concentration
Authors: Treuner-Lange A , Kuhn A , Dürre P
Published: November 2016

The complete sequence of the kdp gene region of Clostridium acetobutylicum has been determined. This part of the chromosome comprises two small open reading frames (orfZ and orfY), putatively encoding hydrophobic peptides, and the genes kdpA, kdpB, kdpC, and kdpX, followed by an operon encoding a pair of sensor-effector regulatory proteins (KdpD and KdpE). Except for orfZ, orfY, and kdpX, all genes showed significant homology to the kdp genes of Escherichia coli, encoding a high-affinity potassium transport ATPase and its regulators. The complete genome sequence of Synechocystis sp. strain PCC 6803 and a recently published part of the Mycobacterium tuberculosis genome indicate the existence of a kdp system in these organisms as well, but all three systems comprise neither a second orf upstream of kdpA nor an additional kdpX gene. Expression of the clostridial kdp genes, including the unique kdpX gene, was found to be inducible by low potassium concentrations. A transcription start point could be mapped upstream of orfZ. A promoter upstream of kdpD was active only under noninducing conditions. Lowering the potassium content of the medium led to formation of a common transcript (orfZYkdpABCXDE), with a putative internal RNase E recognition site, which could be responsible for the instability of the common transcript. Except for the two small peptides, all gene products could be detected in in vitro transcription-translation experiments.
Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilis
Authors: Yoshida K I , Aoyama D , Ishio I , Shibayama T , Fujita Y
Published: November 2016

Previous determination of the nucleotide sequence of the iol region of the Bacillus subtilis genome allowed us to predict the structure of the iol operon for myo-inositol catabolism, consisting of 10 iol genes (iolA to iouJ); iolG corresponds to idh, encoding myo-inositol 2-dehydrogenase (Idh). Primer extension analysis suggested that an inositol-inducible promoter for the iol operon (iol promoter) might be a promoter-like sequence in the 5' region of iolA, which is probably recognized by sigmaA. S1 nuclease analysis implied that a rho-independent terminator-like structure in the 3' region of iolJ might be a terminator for iol transcription. Disruption of the iol promoter prevented synthesis of the iol transcript as well as that of Idh, implying that the iol operon is most probably transcribed as an 11.5-kb mRNA containing the 10 iol genes. Immediately upstream of the iol operon, two genes (iolR and iolS) with divergent orientations to the iol operon were found. Disruption of iolR (but not iolS) caused constitutive synthesis of the iol transcript and Idh, indicating that the iolR gene encodes a transcription-negative regulator (presumably a repressor) for the iol operon. Northern and S1 nuclease analyses revealed that the iolRS genes were cotranscribed from another inositol-inducible promoter, which is probably recognized by sigmaA. The promoter assignments of the iol and iolRS operons were confirmed in vivo with a lacZ fusion integrated into the amyE locus.
A rubrerythrin operon and nigerythrin gene in Desulfovibrio vulgaris (Hildenborough)
Authors: Lumppio H L , Shenvi N V , Garg R P , Summers A O , Kurtz D M
Published: November 2016

Rubrerythrin is a nonheme iron protein of unknown function isolated from Desulfovibrio vulgaris (Hildenborough). We have sequenced a 3.3-kbp Sal1 fragment of D. vulgaris chromosomal DNA containing the rubrerythrin gene, rbr, identified additional open reading frames (ORFs) adjacent to rbr, and shown that these ORFs are part of a transcriptional unit containing rbr. One ORF, designated fur, lies just upstream of rbr and encodes a 128-amino-acid-residue protein which shows homology to Fur (ferric uptake regulatory) proteins from other purple bacteria. The other ORF, designated rdl, lies just downstream of rbr and encodes a 74-residue protein with significant sequence homology to rubredoxins but with a different number and spacing of cysteine residues. Overexpression of rdl in Escherichia coli yielded a protein, Rdl, which has spectroscopic properties and iron content consistent with one Fe3+(SCys)4 site per polypeptide but is clearly distinct from both rubrerythrin and a related protein, nigerythrin. Northern analysis indicated that fur, rbr, and rdl were each present on a transcript of 1.3 kb; i.e., these three genes are cotranscribed. Because D. vulgaris nigerythrin appears to be closely related to rubrerythrin, and its function is also unknown, we cloned and sequenced the gene encoding nigerythrin, ngr. The amino acid sequence of nigerythrin is 33% identical to that of rubrerythrin, and all residues which furnish iron ligands to both the FeS4 and diiron-oxo sites in rubrerythrin are conserved in nigerythrin. Despite the close resemblance of these two proteins, ngr was found to be no closer than 7 kb to rbr on the D. vulgaris chromosome, and Northern analysis showed that, in contrast to rbr, ngr is not cotranscribed with other genes. Possible redox-linked functions for rubrerythrin and nigerythrin in iron homeostasis are proposed.
Dual multimodular class A penicillin-binding proteins in Mycobacterium leprae
Authors: Lepage S , Dubois P , Ghosh T K , Joris B , Mahapatra S , Kundu M , Basu J , Chakrabarti P , Cole S T , Nguyen-Distèche M , Ghuysen J M
Published: November 2016

The ponA gene of cosmid L222 of the Mycobacterium leprae genome library encodes a multimodular class A penicillin-binding protein (PBP), PBP1. The PBP, labelled with a polyhistidine sequence, has been produced in Escherichia coli, extracted from the membranes with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and purified by Ni2(+)-nitrilotriacetic acid-agarose chromatography. In contrast to the pon1-encoded class A PBP1, PBP1 undergoes denaturation at temperatures higher than 25 degrees C, it catalyzes acyl transfer reactions on properly structured thiolesters, and it binds penicillin with high affinity.
Transcription of Staphylococcus aureus fibronectin binding protein genes is negatively regulated by agr and an agr-independent mechanism
Authors: Saravia-Otten P , Müller H P , Arvidson S
Published: November 2016

The production of cell surface proteins in Staphylococcus aureus is generally down-regulated in the postexponential growth phase by the global regulator agr. The effector of this regulation is the RNAIII molecule, which is encoded within the agr locus. RNAIII seems to regulate most target genes at the level of transcription, but it also has an effect on the translation of some genes. To study the role of agr on the expression of fibronectin binding proteins (FnBPs), we investigated the transcription and translation of fnb genes in agr mutant strain WA250 and its parent strain, 8325-4. The results show that fnb genes are negatively regulated by agr and also by an agr-independent mechanism that restricts fnb mRNA synthesis to the early exponential phase of growth. Transcription and Western blot analysis of cell-associated FnBPs demonstrated that synthesis of both FnBPA and FnBPB in the wild-type and agr mutant strains took place preferentially during the first hour of growth and rapidly decreased after the second hour. We also confirmed previous results showing that the agr mutant strain has an increased capacity to bind fibronectin compared to its parent agr+ strain. However, while the concentrations of fnb mRNAs and proteins differed by a factor of 16 between the strains, the difference in fibronectin binding was only twofold, indicating that the binding of fibronectin to the bacteria is not proportional to the amount of FnBPs on their surface.