bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1
Authors: Park S M , Lu C D , Abdelal A T
Published: November 2016

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.
The femR315 gene from Staphylococcus aureus, the interruption of which results in reduced methicillin resistance, encodes a phosphoglucosamine mutase
Authors: Jolly L , Wu S , van Heijenoort J , de Lencastre H , Mengin-Lecreulx D , Tomasz A
Published: November 2016

The femR315 gene was recently identified by Tn551 insertional mutagenesis as one of the new auxiliary genes, the alteration of which resulted in a drastically reduced methicillin resistance of the Staphylococcus aureus strain COL. femR315 (also known as femD) theoretically encoded a protein of 451 amino acids showing significant amino acid sequence homology with phosphoglucomutases and similar enzymes catalyzing the isomerization of hexoses and hexosamine phosphates (S. Wu, H. de Lencastre, A. Sali, and A. Tomasz, Microb. Drug Resist. 2:277-286, 1996). We describe here the overproduction and purification of the FemR315 protein as well as its identification as the phosphoglucosamine mutase which catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, the first step in the reaction sequence leading to the essential peptidoglycan precursor UDP-N-acetylglucosamine. On the basis of these findings, we propose to change the names femR315 and femD to the functionally more appropriate name glmM.
An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport
Authors: Belitsky B R , Gustafsson M C , Sonenshein A L , Von Wachenfeldt C
Published: November 2016

The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.
Environmental signals triggering methylenomycin production by Streptomyces coelicolor A3(2)
Authors: Hayes A , Hobbs G , Smith C P , Oliver S G , Butler P R
Published: November 2016

Methylenomycin production by Streptomyces coelicolor A3(2) may be triggered by either of two environmental signals: alanine growth-rate-limiting conditions and/or an acidic pH shock. The production of this SCP1-encoded antibiotic was studied by using batch and chemostat cultures. Batch cultures indicated a role for both nutritional status and culture pH in its regulation. Steady-state methylenomycin production and transcription of an mmy gene under alanine but not glucose growth-rate-limiting conditions was demonstrated in chemostat culture. Transient mmy expression and methylenomycin production occurred following an acidic pH shock. This stimulation of methylenomycin production occurred independently of the nutritional status of the growth environment. Antibiotic production was partially suppressed under alanine compared with glucose growth-rate-limiting conditions following the acidic pH shock. A low specific growth rate was a prerequisite for both steady-state and transient production of methylenomycin.
A transcriptional activator, FleQ, regulates mucin adhesion and flagellar gene expression in Pseudomonas aeruginosa in a cascade manner
Authors: Arora S K , Ritchings B W , Almira E C , Lory S , Ramphal R
Published: November 2016

Previous work has demonstrated that fleR, the gene for a transcriptional activator belonging to the NtrC subfamily of response regulators, is involved in the regulation of mucin adhesion and flagellar expression by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional regulator which also regulates mucin adhesion and motility in this organism. The complete nucleotide sequence of the fleQ gene was determined on both DNA strands, and an open reading frame (ORF) consisting of 1,493 nucleotides was identified. This ORF coded for a gene product of predicted molecular weight, as confirmed by the overexpression of the fleQ gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the fleSR operon on the 3' end. FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, which work in concert with the alternate sigma factor RpoN (sigma54) to activate transcription. However, FleQ lacks the residues corresponding to Asp-54 and Lys-104 of the NtrC protein which are conserved in most of the members belonging to this subfamily of regulators. In addition, unlike some of the other transcriptional activators of this group, FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fleQ gene abolished mucin adhesion and motility of P. aeruginosa PAK and PAK-NP. Both of these functions were regained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is also necessary for the same process, suggested that these regulators may interact in some way. We therefore examined the regulation of the fleSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other hand, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, adds another level of regulation to motility and adhesion in P. aeruginosa, above that of fleSR. We therefore propose the existence of a regulatory cascade which consists of at least two transcriptional regulators, FleQ and FleR, in the control of motility and adhesion in P. aeruginosa.
Properties of the phosphorylation reaction catalyzed by SpoIIAB that help to regulate sporulation of Bacillus subtilis
Authors: Najafi S M , Harris D A , Yudkin M D
Published: November 2016

Phosphorylation of SpoIIAA on Ser-58 catalyzed by SpoIIAB is important in the regulation of sporulation of Bacillus subtilis. Nucleotide binding experiments showed that the affinity of SpoIIAB for ATP was greatly increased in the presence of SpoIIAA or a mutant SpoIIAA in which Ser-58 had been changed to alanine. Study of the phosphorylation reaction showed that the Km for ATP and the Ki for ADP were both about 1 microM. The kinetics of phosphorylation of SpoIIAA by SpoIIAB were biphasic, comprising a rapid phase (leading to phosphorylation of 1 mol of SpoIIAA/mol of SpoIIAB) followed by a slower, steady-state phase. In the steady state, the rate-determining step proved to be the dissociation of a SpoIIAB-ADP complex. The rate of this dissociation was not affected significantly by changes in the concentration of ATP.
Genomic fluidity of Bordetella pertussis assessed by a new method for chromosomal mapping
Authors: Stibitz S , Yang M S
Published: November 2016

The genomic organization of Bordetella pertussis strains has been examined by using a new method. This method does not depend on the prior determination of a restriction map of the bacterial chromosome but is based on the ability to measure directly the distance between two genes. This is accomplished through the integration at each gene of a suicide vector containing a cleavage site for the intron-encoded endonuclease I-SceI, which is not otherwise found in the chromosome. Integration is mediated by homologous recombination between the chromosomal and cloned plasmid copies of a gene of interest. Digestion with I-SceI gives rise to a fragment the size of which represents the distance between the two genes. Multiple pairwise determinations within a set of genes provide sufficient information to derive a map of the relative gene positions. Mapping a set of 11 to 13 genes for five strains of B. pertussis and one strain of B. parapertussis revealed extensive divergence of gene order between B. pertussis Tohama I, B. pertussis 18-323, and B. parapertussis ATCC 15311. Less extensive divergence of gene order was observed between B. pertussis Tohama I and B. pertussis Tohama III, BP165, and Wellcome 28, with most of the observed differences explainable by large inversions.
The bla gene of the cephamycin cluster of Streptomyces clavuligerus encodes a class A beta-lactamase of low enzymatic activity
Authors: Pérez-Llarena F , Martín J F , Galleni M , Coque J J , Fuente J L , Frère J M , Liras P
Published: November 2016

A gene (bla) encoding a beta-lactamase is present in the cephamycin gene cluster of Streptomyces clavuligerus, the strain producing clavulanic acid and a beta-lactamase inhibitory protein. The bla gene is located 5.1 kb downstream from and in the opposite orientation to cefE, encoding the deacetoxycephalosporin C synthase. The bla gene encodes a 332-residue protein (Mr, 35,218), similar to other class A beta-lactamases produced by actinomycetes. Modification (to SDG) of the SDN conserved motif of class A beta-lactamases as well as of amino acids in otherwise conserved regions in the molecule may explain the low penicillinase and cephalosporinase activities of the protein. The beta-lactamase has been purified to homogeneity and found to bind [3H]benzylpenicillin, a result reflecting a rate-limiting deacylation step. Nucleotide sequences homologous to bla were found in all tested cephamycin producers, but several other Streptomyces species which produce a beta-lactamase do not contain genes for beta-lactam antibiotic biosynthesis.
Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli
Authors: Deloche O , Kelley W L , Georgopoulos C
Published: November 2016

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.
Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli
Authors: González-Flecha B , Demple B
Published: November 2016

The exponential phase of aerobic growth is associated with risk of endogenous oxidative stress in which cells need to cope with an approximately 10-fold increase in the rate of H2O2 generation. We addressed this issue by studying the regulation of the intracellular concentration of H2O2 in aerobically growing Escherichia coli. Intracellular H2O2 was kept at an almost constant steady-state value of approximately 0.2 microM (variation, less than twofold) over a broad range of cell densities in rich medium. This regulation was achieved in part by a transient increase in the OxyR-dependent transcription of the catalase gene katG (monitored by using a katG::lacZ operon fusion) during exponential growth, directly correlated with the increased rate of H2O2 generation. The OxyR-regulated alkyl hydroperoxide reductase encoded by ahpFC did not detectably affect H2O2 or catalase activity levels. Induction of katG, ahpFC, and perhaps other genes prevented the accumulation of oxidatively modified lipids but may not have protected DNA: the spontaneous mutation rate was significantly increased in both wild-type and delta(oxy)R strains during exponential growth compared to that in these strains during lag or stationary phases. Strains lacking oxyR showed throughout growth an 8- to 10-fold-higher frequency of spontaneous mutation than was seen for wild-type bacteria. The ahpdelta5 allele also had a mutator effect half of that of delta(oxy)R in exponential and stationary phases and equal to that of deltaoxyR in lag phase, perhaps by affecting organic peroxide levels. These results show that oxyR-regulated catalase expression is not solely an emergency response of E. coli to environmental oxidative stress, but also that it mediates a homeostatic regulation of the H2O2 produced by normal aerobic metabolism. The activation of the oxyR regulon in this process occurs at much lower levels of H2O2 (approximately 10(-7)M) than those reported for oxyR activation by exogenous H2O2 (approximately 10(-5) M).
Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7
Authors: Iwabuchi T , Harayama S
Published: November 2016

2-Carboxybenzaldehyde dehydrogenase from the phenanthrene-degrading bacterium Nocardioides sp. strain KP7 was purified and characterized. The purified enzyme had a molecular mass of 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 205 kDa by gel filtration chromatography. Thus, the homotetramer of the 53-kDa subunit constituted an active enzyme. The apparent Km and kcat values of this enzyme for 2-carboxybenzaldehyde were 100 microM and 39 s(-1), respectively, and those for NAD+ were 83 microM and 32 s(-1), respectively. The structural gene for this enzyme was cloned and sequenced. The length of the gene was 1,455 bp. The nucleotide sequence of the 10,279 bp of DNA around the gene for 2-carboxybenzaldehyde dehydrogenase was also determined, and seven open reading frames were found in this DNA region. These were the genes for 1-hydroxy-2-naphthoate dioxygenase (phdI) and trans-2'-carboxybenzalpyruvate aldolase (phdJ), orf1, the gene for 2-carboxybenzaldehyde dehydrogenase (phdK), orf2/orf3, and orf4. The amino acid sequence of the orf1 product was similar to that of the aromatic hydrocarbon transporter gene (pcaK) in Pseudomonas putida PRS2000. The amino acid sequence of the orf4 product revealed a similarity to cytochrome P-450 proteins. The region between phdK and orf4 encoded orf2 and orf3 on different strands. The amino acid sequences of the orf2 and orf3 products exhibited no significant similarity to the reported sequences in protein databases.
Regulation of upp expression in Escherichia coli by UTP-sensitive selection of transcriptional start sites coupled with UTP-dependent reiterative transcription
Authors: Tu A H , Turnbough C L
Published: November 2016

Expression of the upp gene of Escherichia coli, which encodes the pyrimidine salvage enzyme uracil phosphoribosyltransferase, is negatively regulated by pyrimidine availability. In this study, we demonstrate that this regulation occurs mainly by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be productively elongated. The upp initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand). Transcription is initiated primarily at the first two bases in this sequence, designated G6 and A7 (counting from the promoter -10 region). High intracellular levels of UTP favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive nucleotide addition) within the run of T residues in the initially transcribed region. The resulting AUUUUn (where n = 1 to >50) transcripts are not extended to include downstream upp sequences. In contrast, low intracellular levels of UTP strongly favor initiation at position G6, which results in transcripts that generally do not engage in reiterative transcription and thus can be normally elongated. This mechanism ensures that high levels of uracil phosphoribosyltransferase are produced only under conditions of pyrimidine limitation. The mechanisms that account for UTP-sensitive start site selection and different fates of upp transcripts, as well as the general use of UTP-dependent reiterative transcription in gene regulation, are discussed in detail.
Photoresponses of the purple nonsulfur bacteria Rhodospirillum centenum and Rhodobacter sphaeroides
Authors: Sackett M J , Armitage J P , Sherwood E E , Pitta T P
Published: November 2016

We have measured the photoresponse of two purple nonsulfur bacteria, Rhodobacter sphaeroides and Rhodospirillum centenum, under defined conditions in a light beam propagating at 90 degrees to the optical axis of the microscope. This beam presented cells with a steep gradient of intensity perpendicular to the direction of propagation and a shallow gradient in the direction of light propagation. R. centenum, a species that reverses to change direction, accumulated in the light beam, as expected for a "scotophobic" response, while R. sphaeroides, which stops rather than reverses, accumulated outside the light beam. We also compared the behavior of liquid-grown R. centenum, which swims by using a single polar flagellum, to that of surface-grown R. centenum, which swarms over agar by using many lateral flagella and has been shown to move as colonies toward specific wavelengths of light. When suspended in liquid medium, both liquid- and surface-grown R. centenum showed similar responses to the light gradient. In all cases, free-swimming cells responded to the steep gradient of intensity but not to the shallow gradient, indicating they cannot sense the direction of light propagation but only its intensity. In a control experiment, the known phototactic alga Chlamydamonas reinhardtii was shown to swim in the direction of light propagation.
The tigA gene is a transcriptional fusion of glycolytic genes encoding triose-phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase in oomycota
Authors: Unkles S E , Logsdon J M , Robison K , Kinghorn J R , Duncan J M
Published: November 2016

Genes encoding triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are fused and form a single transcriptional unit (tigA) in Phytophthora species, members of the order Pythiales in the phylum Oomycota. This is the first demonstration of glycolytic gene fusion in eukaryotes and the first case of a TPI-GAPDH fusion in any organism. The tigA gene from Phytophthora infestans has a typical Oomycota transcriptional start point consensus sequence and, in common with most Phytophthora genes, has no introns. Furthermore, Southern and PCR analyses suggest that the same organization exists in other closely related genera, such as Pythium, from the same order (Oomycota), as well as more distantly related genera, Saprolegnia and Achlya, in the order Saprolegniales. Evidence is provided that in P. infestans, there is at least one other discrete copy of a GAPDH-encoding gene but not of a TPI-encoding gene. Finally, a phylogenetic analysis of TPI does not place Phytophthora within the assemblage of crown eukaryotes and suggests TPI may not be particularly useful for resolving relationships among major eukaryotic groups.
The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains
Authors: Mootz H D , Marahiel M A
Published: November 2016

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.
katGI and katGII encode two different catalases-peroxidases in Mycobacterium fortuitum
Authors: Menéndez M C , Ainsa J A , Martín C , García M J
Published: November 2016

It has been suggested that catalase-peroxidase plays an important role in several aspects of mycobacterial metabolism and is a virulence factor in the main pathogenic mycobacteria. In this investigation, we studied genes encoding for this protein in the fast-growing opportunistic pathogen Mycobacterium fortuitum. Nucleotide sequences of two different catalase-peroxidase genes (katGI and katGII) of M. fortuitum are described. They show only 64% homology at the nucleotide level and 55% identity at the amino acid level, and they are more similar to catalases-peroxidases from different bacteria, including mycobacteria, than to each other. Both proteins were found to be expressed in actively growing M. fortuitum, and both could also be expressed when transformed into Escherichia coli and M. aurum. We detected the presence of a copy of IS6100 in the neighboring region of a katG gene in the M. fortuitum strain in which this element was identified (strain FC1). The influence of each katG gene on isoniazid (isonicotinic acid hydrazide; INH) susceptibility of mycobacteria was checked by using the INH-sensitive M. aurum as the host. Resistance to INH was induced when katGI was transformed into INH-sensitive M. aurum, suggesting that this enzyme contributes to the natural resistance of M. fortuitum to the drug. This is the first report showing two different genes encoding same enzyme activity which are actively expressed within the same mycobacterial strain.
Butyrolactone autoregulator receptor protein (BarA) as a transcriptional regulator in Streptomyces virginiae
Authors: Kinoshita H , Ipposhi H , Okamoto S , Nakano H , Nihira T , Yamada Y
Published: November 2016

BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolides (VBs), a member of the butyrolactone autoregulators of Streptomyces species. Sequencing around the barA gene revealed two novel open reading frames: one upstream, barX, encoding a homolog of AfsA of Streptomyces griseus and another downstream, barB. Northern (RNA) blot analysis for S. virginiae demonstrated that the addition of VB during cultivation switched on the expression of barB. An in vivo expression system in Streptomyces lividans with the use of the xylE reporter gene indicated that BarA in conjunction with VB controlled the barB promoter. Furthermore, the DNA binding ability of BarA was demonstrated in vitro for the first time by means of surface plasmon resonance and a gel-shift assay. Complex formation with VB in vitro resulted in the dissociation of BarA from DNA, thus suggesting that the VB receptor, BarA, is a transcriptional regulator and that the VB signal is transduced to the next step in the signal transduction pathway by modification of the DNA binding ability of BarA.
Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis
Authors: Wyckoff E E , Stoebner J A , Reed K E , Payne S M
Published: November 2016

Vibrio cholerae secretes the catechol siderophore vibriobactin in response to iron limitation. Vibriobactin is structurally similar to enterobactin, the siderophore produced by Escherichia coli, and both organisms produce 2,3-dihydroxybenzoic acid (DHBA) as an intermediate in siderophore biosynthesis. To isolate and characterize V. cholerae genes involved in vibriobactin biosynthesis, we constructed a genomic cosmid bank of V. cholerae DNA and isolated clones that complemented mutations in E. coli enterobactin biosynthesis genes. V. cholerae homologs of entA, entB, entC, entD, and entE were identified on overlapping cosmid clones. Our data indicate that the vibriobactin genes are clustered, like the E. coli enterobactin genes, but the organization of the genes within these clusters is different. In this paper, we present the organization and sequences of genes involved in the synthesis and activation of DHBA. In addition, a V. cholerae strain with a chromosomal mutation in vibA was constructed by marker exchange. This strain was unable to produce vibriobactin or DHBA, confirming that in V. cholerae VibA catalyzes an early step in vibriobactin biosynthesis.
Unusual structure of the tonB-exb DNA region of Xanthomonas campestris pv. campestris: tonB, exbB, and exbD1 are essential for ferric iron uptake, but exbD2 is not
Authors: Wiggerich H G , Klauke B , Köplin R , Priefer U B , Pühler A
Published: November 2016

The nucleotide sequence of a 3.6-kb HindIII-SmaI DNA fragment of Xanthomonas campestris pv. campestris revealed four open reading frames which, based on sequence homologies, were designated tonB, exbB, exbD1, and exbD2. Analysis of translational fusions to alkaline phosphatase and beta-galactosidase confirmed that the TonB, ExbB, ExbD1, and ExbD2 proteins are anchored in the cytoplasmic membrane. The TonB protein of X. campestris pv. campestris lacks the conserved (Glu-Pro)n and (Lys-Pro)m repeats but harbors a 13-fold repeat of proline residues. By mutational analysis, the tonB, exbB, and exbD1 genes were shown to be essential for ferric iron import in X. campestris pv. campestris. In contrast, the exbD2 gene is not involved in the uptake of ferric iron.
Regulation of expression of the ethanol dehydrogenase gene (adhE) in Escherichia coli by catabolite repressor activator protein Cra
Authors: Mikulskis A , Aristarkhov A , Lin E C
Published: November 2016

The adhE gene encodes ethanol dehydrogenase and is located at min 27.9 of the Escherichia coli chromosome. Expression of adhE is about 10-fold higher in cells grown anaerobically than in cells grown aerobically and is dependent on both transcriptional and posttranscriptional factors. In this study, a trans-regulatory element repressing adhE expression was characterized by genetic and biochemical approaches. A mutation downregulating adhE expression was mapped at min 2 of the chromosome. DNA sequence analysis revealed a missense mutation in the cra gene, formerly known as fruR. The cra gene encodes a catabolite repressor-activator protein (Cra) involved in the modulation of carbon flow in E. coli. The mutant protein (Cra*) sustained an Arg148-->His substitution causing 1.5- and 3-fold stronger repression of adhE transcription under anaerobic and aerobic conditions, respectively. By contrast, cra null mutants displayed 1.5- and 4-fold increased adhE transcription under those conditions. Disruption of the cra gene did not abolish the anaerobic activation of the adhE gene but diminished it twofold. Cra and Cra* were purified as fusion proteins tagged with an N-terminal 6xHis element. In vitro, both fusion proteins showed binding to the adhE promoter region and to the control fruB promoter region, which is a known Cra target. However, only 6xHis-tagged Cra, and not 6xHis-Cra*, was displaced from the DNA target by the effector, fructose-1-phosphate (F1P), suggesting that the mutant protein is locked in a promoter-binding conformation and is no longer responsive to F1P. We suggest that Cra helps to tighten the control of adhE transcription under aerobic conditions by its repression.
A gene (plsD) from Clostridium butyricum that functionally substitutes for the sn-glycerol-3-phosphate acyltransferase gene (plsB) of Escherichia coli
Authors: Heath R J , Goldfine H , Rock C O
Published: November 2016

The sn-glycerol-3-phosphate acyltransferase (plsB) of Escherichia coli is a key regulatory enzyme that catalyzes the first committed step in phospholipid biosynthesis. We report the initial characterization of a novel gene (termed plsD) from Clostridium butyricum, cloned based on its ability to complement the sn-glycerol-3-phosphate auxotrophic phenotype of a plsB mutant strain of E. coli. Unlike the 83-kDa PlsB acyltransferase from E. coli, the predicted plsD open reading frame encoded a protein of 26.5 kDa. Two regions of strong homology to other lipid acyltransferases, including PlsB and PlsC analogs from mammals, plants, yeast, and bacteria, were identified. PlsD was most closely related to the 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC) gene family but did not complement the growth of plsC(Ts) mutants. An in vivo metabolic labeling experiment using a plsB plsX plsC(Ts) strain of E. coli confirmed that the plsD expression restored the ability of the cells to synthesize 1-acyl-glycerol-3-phosphate. However, glycerol-3-phosphate acyltransferase activity was not detected in vitro in assays using either acyl-acyl carrier protein or acyl coenzyme A as the substrate.
Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence
Authors: Furuya N , Komano T
Published: November 2016

Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region, various deletion, insertion, and substitution mutations were introduced into a 92-bp minimal R64 oriT sequence and their effects on oriT function were investigated. This detailed analysis confirms our previous prediction that the R64 oriT region consists of an oriT core sequence and additional sequences necessary for full oriT activity. The oriT core sequence consists of the repeat A sequence, which is recognized by R64 NikA protein, and the nick region sequence, which is conserved among various origins of transfer and is most probably recognized by NikB protein. The oriT core sequence is sufficient for NikAB-mediated oriT-specific nicking. Furthermore, it was shown that the repeat A sequence is essential for localization to a precise position relative to the nick site for oriT function. This seems to be required for the formation of a functional ternary complex consisting of NikA and NikB proteins and oriT DNA. The repeat B sequence and 8-bp inverted repeat sequences are suggested to be required for the termination of DNA transfer.
Genetic requirements and mutational specificity of the Escherichia coli SOS mutator activity
Authors: Fijalkowska I J , Dunn R L , Schaaper R M
Published: November 2016

To better understand the mechanisms of SOS mutagenesis in the bacterium Escherichia coli, we have undertaken a genetic analysis of the SOS mutator activity. The SOS mutator activity results from constitutive expression of the SOS system in strains carrying a constitutively activated RecA protein (RecA730). We show that the SOS mutator activity is not enhanced in strains containing deficiencies in the uvrABC nucleotide excision-repair system or the xth and nfo base excision-repair systems. Further, recA730-induced errors are shown to be corrected by the MutHLS-dependent mismatch-repair system as efficiently as the corresponding errors in the rec+ background. These results suggest that the SOS mutator activity does not reflect mutagenesis at so-called cryptic lesions but instead represents an amplification of normally occurring DNA polymerase errors. Analysis of the base-pair-substitution mutations induced by recA730 in a mismatch repair-deficient background shows that both transition and transversion errors are amplified, although the effect is much larger for transversions than for transitions. Analysis of the mutator effect in various dnaE strains, including dnaE antimutators, as well as in proofreading-deficient dnaQ (mutD) strains suggests that in recA730 strains, two types of replication errors occur in parallel: (i) normal replication errors that are subject to both exonucleolytic proofreading and dnaE antimutator effects and (ii) recA730-specific errors that are not susceptible to either proofreading or dnaE antimutator effects. The combined data are consistent with a model suggesting that in recA730 cells error-prone replication complexes are assembled at sites where DNA polymerization is temporarily stalled, most likely when a normal polymerase insertion error has created a poorly extendable terminal mismatch. The modified complex forces extension of the mismatch largely at the exclusion of proofreading and polymerase dissociation pathways. SOS mutagenesis targeted at replication-blocking DNA lesions likely proceeds in the same manner.
Nested DNA inversion of Campylobacter fetus S-layer genes is recA dependent
Authors: Dworkin J , Shedd O L , Blaser M J
Published: November 2016

Wild-type strains of Campylobacter fetus are covered by a monomolecular array of surface layer proteins (SLPs) critical for virulence. Each cell possesses eight SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97 to 149 kDa. Variation of SLP expression occurs by a mechanism of nested DNA rearrangement that involves the inversion of a 6.2-kb sapA promoter-containing element alone or together with one or more flanking SLP gene cassettes. The presence of extensive regions of identity flanking the 5' and 3' ends of each SLP gene cassette and of a Chi-like recognition sequence within the 5' region of identity suggests that rearrangement of SLP gene cassettes may occur by a generalized (RecA-dependent) homologous recombination pathway. To explore this possibility, we cloned C. fetus recA and created mutant strains by marker rescue, in which recA is disrupted in either S+ or S- strains. These mutants then were assessed for their abilities to alter SLP expression either in the presence or absence of a complementary shuttle plasmid harboring native recA. In contrast to all previously reported programmed DNA inversion systems, inversion in C. fetus is recA dependent.
cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B
Authors: Ishiguro J , Saitou A , Durán A , Ribas J C
Published: November 2016

The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive) mutant strain was originally isolated as hypersensitive to the spindle poison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J. Ishiguro and Y. Uhara, Jpn. J. Genet. 67:97-109, 1992). We have found that the cps1-12 mutation also confers (i) hypersensitivity to the immunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drug papulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesis both in vivo and in vitro, and (iii) thermosensitive growth at 37 degrees C. Under any of these restrictive treatments, cells swell up and finally lyse. With an osmotic stabilizer, cells do not lyse, but at 37 degrees C they become multiseptated and multibranched. The cps1-12 mutant, grown at a restrictive temperature, showed an increase in sensitivity to lysis by enzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthase activity (173% in the absence of GTP in the reaction), and in cell wall biosynthesis (130% of the wild-type amount). Addition of Ca2+ suppresses hypersensitivity to papulacandin B and septation and branching phenotypes. All of these data suggest a relationship between the cps1+ gene and cell wall synthesis. A DNA fragment containing the cps1+ gene was cloned, and sequence analysis indicated that it encodes a predicted membrane protein of 1,729 amino acids with 15 to 16 transmembrane domains. S. pombe cps1p has overall 55% sequence identity with Fks1p or Fks2p, proposed to be catalytic or associated subunits of Saccharomyces cerevisiae 1,3-beta-D-glucan synthase. Thus, the cps1+ product might be a catalytic or an associated copurifying subunit of the fission yeast 1,3-beta-D-glucan synthase that plays an essential role in cell wall synthesis.
Involvement of CysB and Cbl regulatory proteins in expression of the tauABCD operon and other sulfate starvation-inducible genes in Escherichia coli
Authors: van der Ploeg J R , Iwanicka-Nowicka R , Kertesz M A , Leisinger T , Hryniewicz M M
Published: November 2016

Starvation for sulfate results in increased synthesis of several proteins in Escherichia coli. Among these Ssi (sulfate starvation-induced) proteins are the products of the tauABCD genes, which are required for utilization of taurine as sulfur source for growth. In this study, the role of the cbl gene in expression of tauABCD and other ssi genes was investigated. The protein encoded by cbl shows high sequence similarity to CysB, the LysR-type transcriptional activator of the genes involved in cysteine biosynthesis. Strain EC2541, which contains an internal deletion in cbl, was unable to utilize taurine and other aliphatic sulfonates as sulfur sources. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many of the Ssi proteins were not synthesized in EC2541. Expression of a translational tauD'-'lacZ fusion required the presence of both cbl and cysB. The interactions of CysB and Cbl with the promoter region of tauABCD were studied by using gel mobility shift experiments and DNase I footprinting. CysB occupied multiple binding sites, whereas Cbl occupied only one site from 112 to 68 bp upstream of the transcription start site. Acetylserine, the inducer of transcription of CysB-regulated genes, stimulated binding of CysB but not of Cbl. Sulfate had no effect on binding of both proteins to the tauABCD promoter region. These results indicate that Cbl is a transcription factor for genes required for sulfonate-sulfur utilization and maybe for other genes whose expression is induced by sulfate starvation.
Cloning, functional analysis, and transcriptional regulation of the Bacillus subtilis araE gene involved in L-arabinose utilization
Authors: Sá-Nogueira I , Ramos S S
Published: November 2016

The Bacillus subtilis araR locus (mapped at about 294 degrees on the genetic map) comprises two open reading frames with divergently arranged promoters, the regulatory gene, araR, encoding a repressor, and a partially cloned gene, termed araE by analogy to the Escherichia coli L-arabinose permease gene. Here, we report the cloning and sequencing of the entire araE gene encoding a 50.4-kDa polypeptide. The araE gene is monocistronic (as determined by Northern blot analysis), and its putative product is very similar to a number of prokaryotic proton-linked monosaccharide transporters (the group I family of membrane transport proteins). Insertional inactivation of the araE gene leads to a conditional Ara- phenotype dependent on the concentration of L-arabinose in the medium. Therefore, we assume that araE encodes a permease involved in L-arabinose transport into the cell. The araE promoter region contains -10 and -35 regions (as determined by primer extension analysis) very similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigmaA, and the -35 region of the transcription start point for araE is located 2 bp from the -35 region of the araR gene. Transcriptional studies demonstrated that the expression from the araE promoter is induced by L-arabinose, repressed by glucose, and negatively regulated by AraR. These observations are consistent with a model according to which in the absence of L-arabinose, AraR binds to a site(s) within the araE/araR promoter, preventing transcription from the araE promoter and simultaneously limiting the frequency of initiation from its own promoter; the addition of L-arabinose will allow transcription from the araE promoter and increase the frequency of initiation from the araR promoter.
Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression
Authors: Yang C , Kaplan H B
Published: November 2016

Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene. The wild-type sasS gene was cloned and sequenced. This gene is predicted to encode a sensor histidine protein kinase that appears to be a key element in the transduction of starvation and cell density inputs. The sasS null mutants express 4521 at a basal level, form defective fruiting bodies, and exhibit reduced sporulation efficiencies. These data indicate that the wild-type sasS gene product functions as a positive regulator of 4521 expression and participates in M. xanthus development. The N terminus of SasS is predicted to contain two transmembrane domains that would locate the protein to the cytoplasmic membrane. The sasB7 mutation, an E139K missense mutation, maps to the predicted N-terminal periplasmic region. The C terminus of SasS contains all of the conserved residues typical of the sensor histidine protein kinases. SasS is predicted to be the sensor protein in a two-component system that integrates information required for M. xanthus developmental gene expression.
Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12
Authors: Beary T P , Braymer H D , Achberger E C
Published: November 2016

The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.
Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor
Authors: Bukhalid R A , Loria R
Published: November 2016

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.