bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular evolutionary genetics of the cattle-adapted serovar Salmonella dublin
Authors: Selander R K , Smith N H , Li J , Beltran P , Ferris K E , Kopecko D J , Rubin F A
Published: November 2016

An electrophoretic analysis of allelic variation at 24 enzyme loci among 170 isolates of the serovar Salmonella dublin (serotype 1,9,12[Vi]:g,p:-) identified three electrophoretic types (Du 1, Du 3, and Du 4), marking three closely related clones, one of which (Du 1) is globally distributed and was represented by 95% of the randomly selected isolates. All but 1 of 114 nonmotile isolates of serotype 1,9,12:-:- recovered from cattle and swine in the United States were genotypically Du 1. The virulence capsular polysaccharide (Vi antigen) is confined to clone Du 3, which apparently is limited in distribution to France and Great Britain. For all 29 isolates of Du 3, positive signals were detected when genomic DNA was hybridized with a probe specific for the ViaB region, which contains the structurally determinant genes for the Vi antigen; and 23 of these isolates had been serologically typed as Vi positive. In contrast, all 30 isolates of Du 1 tested with the ViaB probe were negative. These findings strongly suggest that the ViaB genes were recently acquired by S. dublin via horizontal transfer and additive recombination. The clones of S. dublin are closely similar to the globally predominant clone (En 1) of Salmonella enteritidis (serotype 1,9,12:g,m:-) in both multilocus enzyme genotype and nucleotide sequence of the fliC gene encoding phase 1 flagellin. Comparative sequencing of fliC has revealed the molecular genetic basis for expression of the p and m flagellar epitopes by which these serovars are distinguished in the Kauffmann-White serological scheme of classification.
Interaction between ribulose 1,5-bisphosphate carboxylase/oxygenase activity and the ammonia assimilatory system of Rhodobacter sphaeroides
Authors: Wang X , Tabita F R
Published: November 2016

The levels of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Rhodobacter sphaeroides were found to depend on the concentration of ammonia supplied to photolithoautotrophically grown cultures. Under conditions in which the cells rapidly depleted the available ammonia, the level of in situ RubisCO activity decreased to less than 5% maximum activity; even at its maximum level under these conditions, the RubisCO activity was only 5% of the activity obtained from cultures supplied with saturating levels of ammonia. When cells were incubated with somewhat higher but not saturating amounts of ammonia, in situ RubisCO activity decreased immediately after the cells depleted the cultures of ammonia. The decrease in activity was not due to any detectable degradation of RubisCO protein, indicative of some mechanism to regulate the activity of the enzyme in response to the intracellular levels of assimilated ammonia. Furthermore, under conditions optimum for RubisCO inactivation, in situ RubisCO activity in permeabilized whole cells greatly exceeded the levels of enzymatic activity determined in vitro in cell extracts. Blockage of ammonia assimilation by inhibition of glutamine synthetase with methionine sulfoximine prevented the recovery of form I RubisCO from pyruvate-mediated inactivation, suggesting the presence of regulatory mechanisms common to both CO2 fixation and ammonia assimilation.
Interaction of inactivated and active ribulose 1,5-bisphosphate carboxylase/oxygenase of Rhodobacter sphaeroides with nucleotides and the chaperonin 60 (GroEL) protein
Authors: Wang X , Tabita F R
Published: November 2016

Purified inactivated form I ribulose 1,5-bisphosphate carboxylase/oxygenase (form I RubisCO) of Rhodobacter sphaeroides was activated by ATP and, to some extent, by other adenylates and nucleotides. Reactivation in the presence of ATP occurred by a time-dependent and concentration-dependent process which appeared to be irreversible. The carbamylated form of inactivated form I RubisCO was less susceptible to ATP-mediated reactivation than the uncarbamylated inactivated enzyme. In some cases, ATP analogs could mimic the reactivation process; one analog, adenylyl(beta, gamma-methylene)-diphosphonate, was found to partially block ATP-mediated reactivation but could not block reactivation induced by Mg(II). Concomitant with the recovery of enzymatic activity, the migration of the inactivated form I RubisCO on nondenaturing and sodium dodecyl sulfate gels changed from a pattern that was characteristic of inactivated enzyme to a pattern that was identical to that of the active protein. It was further found that discrete proportions of active enzyme and the chaperonin 60 protein of R. sphaeroides aggregated in the presence of ATP. The form I RubisCO is thus proposed to contain a specific ATP-binding site that may contribute to both the regulation of activity and the assembly of active enzyme.
Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium
Authors: Lochmeyer C , Koch J , Fuchs G
Published: November 2016

The enzymes catalyzing the initial reactions in the anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) were studied with a denitrifying Pseudomonas sp. anaerobically grown with 2-aminobenzoate and nitrate as the sole carbon and energy sources. Cells grown on 2-aminobenzoate are simultaneously adapted to growth with benzoate, whereas cells grown on benzoate degrade 2-aminobenzoate several times less efficiently than benzoate. Evidence for a new reductive pathway of aromatic metabolism and for four enzymes catalyzing the initial steps is presented. The organism contains 2-aminobenzoate-coenzyme A ligase (2-aminobenzoate-CoA ligase), which forms 2-aminobenzoyl-CoA. 2-Aminobenzoyl-CoA is then reductively deaminated to benzoyl-CoA by an oxygen-sensitive enzyme, 2-aminobenzoyl-CoA reductase (deaminating), which requires a low potential reductant [Ti(III)]. The specific activity is 15 nmol of 2-aminobenzoyl-CoA reduced min-1 mg-1 of protein at an optimal pH of 7. The two enzymes are induced by the substrate under anaerobic conditions only. Benzoyl-CoA is further converted in vitro by reduction with Ti(III) to six products; the same products are formed when benzoyl-CoA or 2-aminobenzoyl-CoA is incubated under reducing conditions. Two of them were identified preliminarily. One product is cyclohex-1-enecarboxyl-CoA, the other is trans-2-hydroxycyclohexane-carboxyl-CoA. The complex transformation of benzoyl-CoA is ascribed to at least two enzymes, benzoyl-CoA reductase (aromatic ring reducing) and cyclohex-1-enecarboxyl-CoA hydratase. The reduction of benzoyl-CoA to alicyclic compounds is catalyzed by extracts from cells grown anaerobically on either 2-aminobenzoate or benzoate at almost the same rate (10 to 15 nmol min-1 mg-1 of protein). In contrast, extracts from cells grown anaerobically on acetate or grown aerobically on benzoate or 2-aminobenzoate are inactive. This suggests a sequential induction of the enzymes.
The Streptomyces glaucescens TcmR protein represses transcription of the divergently oriented tcmR and tcmA genes by binding to an intergenic operator region
Authors: Guilfoile P G , Hutchinson C R
Published: November 2016

Preliminary evidence has been presented by Guilfoile and Hutchinson (J. Bacteriol. 174:3651-3658, 1992) suggesting that the Streptomyces glaucescens TcmR protein is a transcriptional repressor. Here, we extend that work by showing that transcription of the S. glaucescens tcmA gene is inducible by tetracenomycin C and that inactivation of the tcmR gene results in constitutive transcription of the tcmA gene. Gel retardation studies show that the TcmR protein binds to the tcmA-tcmR intergenic region in vitro and that this binding is inhibited by tetracenomycin C. Footprinting experiments demonstrate that the TcmR protein binds to an operator region that encompasses both the tcmA and the tcmR promoters. This genetic and biochemical evidence strongly supports the model of the TcmR protein acting as a repressor in inhibiting transcription of both the tcmA and the tcmR genes, in much the same way that TetR from Tn10 inhibits transcription of tetA and tetR.
Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267
Authors: Rosenstein R , Peschel A , Wieland B , Götz F
Published: November 2016

The arsenate, arsenite, and antimonite resistance region of the Staphylococcus xylosus plasmid pSX267 was subcloned in Staphylococcus carnosus. The sequenced DNA region revealed three consecutive open reading frames, named arsR, arsB, and arsC. Expression studies in Escherichia coli with the bacteriophage T7 RNA polymerase-promoter system yielded three polypeptides with apparent molecular weights of 8,000, 35,000, and 15,000, which very likely correspond to ArsR, ArsB, and ArsC, respectively. ArsB was distinguished by its overall hydrophobic character, suggesting a membrane association. The arsenate, arsenite, and antimonite resistance was shown to be inducible by all three heavy metal ions. Inactivation of the first gene, arsR, resulted in constitutive expression of resistance. Similar results were obtained with transcriptional fusions of various portions of the ars genes with a lipase reporter gene, indicating a function of ArsR as a negative regulator of a putative promoter in front of arsR. The inactivation of arsR also resulted in reduction of resistance to arsenite and antimonite, while arsenate resistance was unaffected. The three ars genes conferred arsenite resistance in E. coli and arsenite as well as arsenate resistance in Bacillus subtilis.
Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae
Authors: Butterton J R , Stoebner J A , Payne S M , Calderwood S B
Published: November 2016

A 74-kDa iron-regulated outer membrane protein of Vibrio cholerae acts as the receptor for the V. cholerae iron-siderophore complex, ferric vibriobactin. MBG14, a mutant of V. cholerae 0395 containing a TnphoA insertion in a gene designated viuA, lacks this 74-kDa outer membrane protein and is unable to bind or utilize exogenous ferric vibriobactin. Introduction of a plasmid containing the complete viuA coding sequence and 513 bp of upstream DNA into MBG14 restored ferric vibriobactin utilization to the mutant. The DNA insert in this plasmid was sequenced, revealing a single open reading frame of 2,061 bp, encoding a deduced protein of 687 amino acids with a predicted molecular mass of 76,417 Da and a predicted initial signal sequence of 37 amino acids. ViuA showed only weak homology to two iron-regulated outer membrane proteins in Escherichia coli, IutA and FecA. Construction of viuA::TnphoA gene fusions allowed study of the regulation of viuA expression by iron. This regulation in E. coli was dependent on the fur gene. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuA message that was negatively regulated by iron at the transcriptional level. Primer extension analysis identified a single transcriptional start site, located 243 bp above the translational start site. The promoter region of viuA contained two interrupted dyad symmetric nucleotide sequences, overlapping the -10 and -35 boxes, each similar to the E. coli Fur binding consensus sequence. Another iron-regulated gene in V. cholerae that is negatively regulated by fur, irgA, requires a positive transcriptional activator (irgB) for expression. However, a strain of V. cholerae mutant in irgB was unaffected in viuA expression. These studies suggest that there is conserved, global coordinate iron regulation in V. cholerae by fur; additional regulatory factors, superimposed upon the fur system, may provide more precise control of individual iron-regulated genes.
Group B streptococcal opacity variants
Authors: Pincus S H , Cole R L , Wessels M R , Corwin M D , Kamanga-Sollo E , Hayes S F , Cieplak W , Swanson J
Published: November 2016

Colony opacity variants were detected for type III group B streptococci (GBS). Transparent colonies predominate in the parent GBS, with occasional colonies having opaque portions. Two stable opaque variants (1.1 and 1.5) were compared with three transparent clones (1.2, 1.3, and 1.4). All grew well on blood agar and on GC medium, but variant 1.1 failed to grow on Todd-Hewitt medium. Scanning and transmission electron microscopy demonstrated that colony opacity correlated with bacterial aggregation status, with opaque variants forming longer and more organized chains. Opaque-transparent switches were observed in both directions for most variants, with transparent to opaque noted most frequently, but 1.5 did not switch at all. Switching of the opacity phenotype was observed both in vitro and in neonatal mice. Relationships between colony opacity and several cell surface phenomena were explored. (i) Opaque variant 1.1 had two surface proteins (46 and 75 kDa) that were either unique or greatly overexpressed. (ii) Variant 1.1 was deficient in type III polysaccharide, while 1.5 lacked group B antigen. Diminished capsular polysaccharide of variant 1.1 was reflected in reduced negative electrophoretic mobility and in increased buoyant density. (iii) Transparent variant colonies growing closest to a penicillin disk were opaque, but colonial variants did not differ in their sensitivity to penicillin. These data indicate that GBS can exist in both opaque and transparent forms, with opaque appearance occurring by multiple routes. Opaque variants grow poorly on Todd-Hewitt medium generally used for isolation of GBS, so any possible relationships between opacity variation and pathogenesis of GBS infection are unknown.
Physical map of the linear chromosome of the bacterium Borrelia burgdorferi 212, a causative agent of Lyme disease, and localization of rRNA genes
Authors: Davidson B E , MacDougall J , Saint Girons I
Published: November 2016

The spirochete Borrelia burgdorferi, which causes Lyme disease, and other members of the Borrelia genus are unique among characterized bacteria in having a linear chromosome. A restriction map of the chromosome of B. burgdorferi 212 was constructed by making extensive use of digestions in agarose blocks of restriction endonuclease fragments or chromosomal DNA that had been purified by pulsed-field gel electrophoresis. A total of 47 digestion sites for the enzymes SgrAI, SacII, MluI, BssHII, EagI, SmaI, NaeI, and ApaI were located. In most regions of the map, the gap between sites is 50 kbp or less, and 122 kbp is the largest distance between adjacent sites. The mapping data were consistent with previous conclusions that the B. burgdorferi chromosome is linear. The total size of the B. burgdorferi 212 chromosome was determined to be 946 kbp from the sums of the sizes of SacII, MluI, BssHII, and SmaI fragments, making it one of the smallest known bacterial chromosomes. The rRNA genes were found to be located near the center of the chromosome. One copy of the 16S rRNA gene (rrs) and two copies of the 23S rRNA gene (designated rrlA and rrlB), the latter pair in a tandem repeat, were detected. This particular complement of these two genes has not been reported for another bacterium.
Localization of TraC, a protein involved in assembly of the F conjugative pilus
Authors: Schandel K A , Muller M M , Webster R E
Published: November 2016

TraC is one of the proteins encoded by the F transfer region of the F conjugative plasmid which is required for the assembly of F pilin into the mature F pilus structure. Overproduction of this protein from the plasmid pKAS2, which carries only traC, resulted in the formation of inclusion bodies from which soluble TraC was purified. When small amounts of TraC were produced from pKAS2, the protein was localized to the cytoplasm by using anti-TraC antibodies. Similar analysis of a set of TraC-alkaline phosphatase fusion proteins localized all of these fusion proteins to the cytoplasm. However, when TraC was expressed from the F plasmid, much of it appeared associated with the bacterial membrane fraction. Under these conditions, TraC does not appear to be part of the tip of the F pilus, as neither anti-TraC antibodies nor purified TraC had any effect on the infection of F-containing bacteria by the filamentous bacteriophage f1. These data suggest that TraC is normally associated with the membrane through interactions with other proteins specified by the tra region. This interaction may be via the carboxyl-terminal region of the TraC protein, as a mutant TraC protein containing an Arg-Cys substitution at amino acid 811 exhibits an interaction with the membrane weaker than that of the wild-type protein in the presence of the other Tra proteins.
Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange
Authors: Ferrero R L , Cussac V , Courcoux P , Labigne A
Published: November 2016

Isogenic urease-negative mutants of Helicobacter pylori were constructed by allelic replacement. A region of cloned H. pylori DNA containing the structural urease genes (ureA and ureB) was disrupted by insertion of a mini-Tn3-Km transposon. Electrotransformation of H. pylori cells with kanamycin-ureB-disrupted derivative plasmids resulted in isolation of kanamycin-resistant H. pylori transformants. Competence for electrotransformation appeared to be restricted to certain wild-type H. pylori isolates; only 1 isolate (of 10 tested) was consistently transformed. Two of the kanamycin-resistant H. pylori transformants were further studied and shown to be urease negative. Southern hybridization analyses demonstrated that the urease-negative mutants had been constructed by allelic exchange involving simultaneous replacement of the ureB gene with the kanamycin-ureB-disrupted copy and loss of the vector. Immunoblot studies of whole-cell extracts of the isogenic ureB mutants with anti-H. pylori sera indicated the absence of a polypeptide with an apparent molecular mass of 61 kDa; thus, the mutants no longer synthesized the UreB product. Generation of stable, genetically engineered urease mutants of H. pylori will be useful for addressing the role of urease in the pathogenesis of H. pylori infection.
Influence of attractants and repellents on methyl group turnover on methyl-accepting chemotaxis proteins of Bacillus subtilis and role of CheW
Authors: Hanlon D W , Carpenter P B , Ordal G W
Published: November 2016

The ability of attractants and repellents to affect the turnover of methyl groups on the methyl-accepting chemotaxis proteins (MCPs) was examined for Bacillus subtilis. Attractants were found to cause an increase in the turnover of methyl groups esterified to the MCPs, while repellents caused a decrease. These reactions do not require CheW. However, a cheW null mutant exhibits enhanced turnover in unstimulated cells. Assuming that the turnover of methyl groups on the MCPs reflects a change in the activity of CheA, these results suggest that the activation of CheA via chemoeffector binding at the receptor does not require CheW.
Identification of a protein-binding sequence involved in expression of an esterase gene from Streptomyces scabies
Authors: Babcock M J , McGrew M , Schottel J L
Published: November 2016

Expression of an esterase gene from Streptomyces scabies is regulated by zinc in both Streptomyces scabies and Streptomyces lividans. A specific protein-binding site was identified on an esterase promoter fragment by using an S-30 extract from S. scabies. The location of the protein-binding site was determined by gel shift assays of promoter deletion fragments and by DNase I footprinting analysis. The protein-binding site maps from nucleotides -59 to -81 relative to the start of transcription. An esterase gene construct cloned and expressed in S. lividans was used to assess the importance of the protein-binding site. Deletion of the 23-bp protein-binding site resulted in a 10-fold decrease in esterase production when cells were grown in zinc-inducing conditions. The protein-binding site may represent a region involved in positive regulation of the S. scabies esterase gene.
TmrB protein, responsible for tunicamycin resistance of Bacillus subtilis, is a novel ATP-binding membrane protein
Authors: Noda Y , Yoda K , Takatsuki A , Yamasaki M
Published: November 2016

tmrB is the gene responsible for tunicamycin resistance in Bacillus subtilis. It is predicted that an increase in tmrB gene expression makes B. subtilis tunicamycin resistant. To examine the tmrB gene product, we produced the tmrB gene product in Escherichia coli by using the tac promoter. TmrB protein was found not only in the cytoplasm fraction but also in the membrane fraction. Although TmrB protein is entirely hydrophilic and has no hydrophobic stretch of amino acids sufficient to span the membrane, its C-terminal 18 amino acids could form an amphiphilic alpha-helix. Breaking this potential alpha-helix by introducing proline residues or a stop codon into this region caused the release of this membrane-bound protein into the cytoplasmic fraction, indicating that the C-terminal 18 residues were essential for membrane binding. On the other hand, TmrB protein has an ATP-binding consensus sequence in the N-terminal region. We have tested whether this sequence actually has the ability to bind ATP by photoaffinity cross-linking with azido-[alpha-32P]ATP. Wild-type protein bound azido-ATP well, but mutants with substitutions in the consensus amino acids were unable to bind azido-ATP. These C-terminal or N-terminal mutant genes were unable to confer tunicamycin resistance on B. subtilis in a multicopy state. It is concluded that TmrB protein is a novel ATP-binding protein which is anchored to the membrane with its C-terminal amphiphilic alpha-helix.
Heat killing of bacterial spores analyzed by differential scanning calorimetry
Authors: Belliveau B H , Beaman T C , Pankratz H S , Gerhardt P
Published: November 2016

Thermograms of the exosporium-lacking dormant spores of Bacillus megaterium ATCC 33729, obtained by differential scanning calorimetry, showed three major irreversible endothermic transitions with peaks at 56, 100, and 114 degrees C and a major irreversible exothermic transition with a peak at 119 degrees C. The 114 degrees C transition was identified with coat proteins, and the 56 degrees C transition was identified with heat inactivation. Thermograms of the germinated spores and vegetative cells were much alike, including an endothermic transition attributable to DNA. The ascending part of the main endothermic 100 degrees C transition in the dormant-spore thermograms corresponded to a first-order reaction and was correlated with spore death; i.e., greater than 99.9% of the spores were killed when the transition peak was reached. The maximum death rate of the dormant spores during calorimetry, calculated from separately measured D and z values, occurred at temperatures above the 73 degrees C onset of thermal denaturation and was equivalent to the maximum inactivation rate calculated for the critical target. Most of the spore killing occurred before the release of most of the dipicolinic acid and other intraprotoplast materials. The exothermic 119 degrees C transition was a consequence of the endothermic 100 degrees C transition and probably represented the aggregation of intraprotoplast spore components. Taken together with prior evidence, the results suggest that a crucial protein is the rate-limiting primary target in the heat killing of dormant bacterial spores.
Transformation of the gram-positive bacterium Clavibacter xyli subsp. cynodontis by electroporation with plasmids from the IncP incompatibility group
Authors: Metzler M C , Zhang Y P , Chen T A
Published: November 2016

We report the transformation of a gram-positive bacterium, Clavibacter xyli subsp. cynodontis, with several plasmids in the IncP incompatibility group from gram-negative bacteria. Our results suggest that IncP plasmids may be transferable to other gram-positive organisms. After optimizing electroporation parameters, we obtained a maximum of 2 x 10(5) transformants per microgram of DNA. The availability of a transformation system for this bacteria will facilitate its use in indirectly expressing beneficial traits in plants.
Cloning, expression, and nucleotide sequence of a mutant glgC gene from Escherichia coli B
Authors: Meyer C R , Ghosh P , Remy E , Preiss J
Published: November 2016

A mutant glgC gene contained in a 10.9-kb PstI fragment was cloned from the Escherichia coli B strain SG5 via colony hybridization by using a wild-type glgC probe. The altered allosteric properties of the expressed ADPglucose synthetase were found to result from the conversion of proline to serine at amino acid residue 295.
Fis plays a role in Tn5 and IS50 transposition
Authors: Weinreich M D , Reznikoff W S
Published: November 2016

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.
Characterization of Escherichia coli glnL mutations affecting nitrogen regulation
Authors: Atkinson M R , Ninfa A J
Published: November 2016

Nitrogen regulator II (NRII), the product of the Escherichia coli glnL (ntrB) gene, regulates the activation of transcription of glnA and the Ntr regulon by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI. Previous results have indicated that under conditions of nitrogen excess, transcriptional activation is prevented by an NRI-phosphate phosphatase activity that is observed when NRII and another signal transduction protein known as PII (the glnB product) interact. The availability of PII for this interaction is controlled by a uridylytransferase/uridylyl-removing enzyme, encoded by glnD, that reversibly modifies PII in response to intracellular signals of nitrogen availability. Here we describe the isolation and characterization of missense mutations in glnL that suppress the Ntr- phenotype resulting from a leaky glnD mutation. The regulation of glnA expression in the pseudorevertants was found to vary from complete insensitivity to ammonia in some strains (GlnC phenotype) to nearly normal regulation by ammonia in other strains. Sequence analysis indicated that in 16 instances suppression was due to point mutations at 14 different sites; 10 different mutations resulting in a variety of phenotypes were identified in a cluster extending from codons 111 to 154 flanking the site of NRII autophosphorylation at His-139. Complementation experiments with multicopy plasmids encoding NRII or PII showed that suppression by GlnC glnL alleles was eliminated upon introduction of the plasmid encoding NRII but was not affected by introduction of the plasmid encoding PII. Conversely, suppression by certain glnL alleles that resulted in regulated expression of glnA was eliminated upon introduction of either the plasmid encoding NRII or that encoding PII. We hypothesize that mutants of the latter type result in a subtle perturbation of the NRII-PII interaction and suggest two possible mechanisms for their effects.
Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii
Authors: Menon A L , Mortenson L E , Robson R L
Published: November 2016

Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.
5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane, a structural component of the modified folate in Sulfolobus solfataricus
Authors: Zhou D , White R H
Published: November 2016

The partial characterization of the modified folate present in Sulfolobus solfataricus has been carried out. Separation of ethanol-water extracts of these cells on a DEAE-Sephadex column led to the isolation of a small amount of intact oxidized cofactor, which, when subjected to reductive cleavage with Zn-HCl, produced 6-methylpterin. This indicated that the modified folate in these cells contained a nonmethylated pterin linked, via a methylene group at the C-6 position of the pterin, to an arylamine, as is found in folate. Oxidative cleavage of intact reduced cofactor produced pterin and a single arylamine. The azo dye derivative of this arylamine was prepared and purified by chromatography on a Bio-Gel P-6 column. The resulting purified compound was shown to be readily hydrolyzed in dilute acid to the azo dye derivative of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypantane, which was, in turn, readily cleaved to 5-(p-aminophenyl)-1,2,3,4- tetrahydroxypentane by Zn-HCl reduction. The stereochemistry of the resulting 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane was shown to be ribo, the same as that of the 5-(p-aminophenyl)-1,2,3,4- tetrahydroxypentane moiety found in methanopterin. The complete arylamine side chain of the modified folate thus contains 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane attached, via an acid-labile bond, to a currently unidentified substituent. The modified folate present in S. solfataricus thus contains structural features common to both folates and methanopterin.
Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium
Authors: Klena J D , Pradel E , Schnaitman C A
Published: November 2016

Analysis of the sequence of a 4.3-kb region downstream of rfaJ revealed four genes. The first two of these, which encode proteins of 27,441 and 32,890 Da, were identified as rfaY and rfaZ by homology of the derived protein sequences of their products to the products of similar genes of Salmonella typhimurium. The amino acid sequences of proteins RfaY and RfaZ showed, respectively, 70 and 72% identity. Genes 3 and 4 were identified as rfaK and rfaL on the basis of size and position, but the derived amino acid sequences of the products of these genes showed very little similarity (about 12% identity) between Escherichia coli K-12 and S. typhimurium. The next gene in the cluster, rfaC, encodes a product which also shows strong protein sequence homology between E. coli K-12 and S. typhimurium, as do the rfaF and rfaD genes which lie beyond it. Thus, the rfa gene cluster appears to consist of two blocks of genes which are conserved flanking a central region of two genes which are not conserved between these species. Although the RfaL protein sequence is not conserved, hydropathy plots of the two RfaL species are nearly identical and indicate that this is a typical integral membrane protein with 10 or more potential transmembrane domains. We noted the similarity of the structure of the rfa gene cluster to that of the rfb gene cluster, which has now been sequenced in several Salmonella serovars. The rfb cluster also contains a gene which lies within a central nonconserved region and encodes an integral membrane protein similar to protein RfaL. We speculate that protein RfaL may interact in a strain- or species-specific way with one or more Rfb proteins in the expression of surface O antigen.
Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene
Authors: McDermott T R , Kahn M L
Published: November 2016

The gene encoding Rhizobium meliloti isocitrate dehydrogenase (ICD) was cloned by complementation of an Escherichia coli icd mutant with an R. meliloti genomic library constructed in pUC18. The complementing DNA was located on a 4.4-kb BamHI fragment. It encoded an ICD that had the same mobility as R. meliloti ICD in nondenaturing polyacrylamide gels. In Western immunoblot analysis, antibodies raised against this protein reacted with R. meliloti ICD but not with E. coli ICD. The complementing DNA fragment was mutated with transposon Tn5 and then exchanged for the wild-type allele by recombination by a novel method that employed the Bacillus subtilis levansucrase gene. No ICD activity was found in the two R. meliloti icd::Tn5 mutants isolated, and the mutants were also found to be glutamate auxotrophs. The mutants formed nodules, but they were completely ineffective. Faster-growing pseudorevertants were isolated from cultures of both R. meliloti icd::Tn5 mutants. In addition to lacking all ICD activity, the pseudorevertants also lacked citrate synthase activity. Nodule formation by these mutants was severely affected, and inoculated plants had only callus structures or small spherical structures.
Structure and regulation of the Yersinia pestis yscBCDEF operon
Authors: Haddix P L , Straley S C
Published: November 2016

We have investigated the physical and genetic structure and regulation of the Yersinia pestis yscBCDEF region, previously called lcrC. DNA sequence analysis showed that this region is homologous to the corresponding part of the ysc locus of Yersinia enterocolitica and suggested that the yscBCDEF cistrons belong to a single operon on the low-calcium response virulence plasmid pCD1. Promoter activity measurements of ysc subclones indicated that yscBCDEF constitutes a suboperon of the larger ysc region by revealing promoter activity in a clone containing the 3' end of yscD, intact yscE and yscF, and part of yscG. These experiments also revealed an additional weak promoter upstream of yscD. Northern (RNA) analysis with a yscD probe showed that operon transcription is thermally induced and downregulated in the presence of Ca2+. Primer extension of operon transcripts suggested that two promoters, a moderate-level constitutive one and a stronger, calcium-downregulated one, control full-length operon transcription at 37 degrees C. Primer extension provided additional support for the proposed designation of a yscBCDEF suboperon by identifying a 5' end within yscF, for which relative abundances in the presence and absence of Ca2+ revealed regulation that is distinct from that for transcripts initiating farther upstream. YscB and YscC were expressed in Escherichia coli by using a high-level transcription system. Attempts to express YscD were only partially successful, but they revealed interesting regulation at the translational level.
Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis
Authors: Snoep J L , Teixeira de Mattos M J , Starrenburg M J , Hugenholtz J
Published: November 2016

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.
The replication initiator operon of promiscuous plasmid RK2 encodes a gene that complements an Escherichia coli mutant defective in single-stranded DNA-binding protein
Authors: Jovanovic O S , Ayres E K , Figurski D H
Published: November 2016

The amino acid sequence of the 13-kDa polypeptide (P116) encoded by the first gene of the trfA operon of IncP plasmid RK2 shows significant similarity to several known single-stranded DNA-binding proteins. We found that unregulated expression of this gene from its natural promoter (trfAp) or induced expression from a strong heterologous promoter (trcp) was sufficient to complement the temperature-sensitive growth phenotype of an Escherichia coli ssb-1 mutant. The RK2 ssb gene is the first example of a plasmid single-stranded DNA-binding protein-encoding gene that is coregulated with replication functions, indicating a possible role in plasmid replication.
The pyocin Sa receptor of Pseudomonas aeruginosa is associated with ferripyoverdin uptake
Authors: Smith A W , Hirst P H , Hughes K , Gensberg K , Govan J R
Published: November 2016

We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity.
Multiple transcripts encoded by the ilvGMEDA gene cluster of Escherichia coli K-12
Authors: Huang F , Coppola G , Calhoun D H
Published: November 2016

We report here that, using Northern (RNA) blots, we identified two relatively stable transcripts of 4.6 and 1.1 kb that correspond to the products of the ilvEDA and ilvE genes and two relatively unstable transcripts of 6.7 and 3.6 kb that correspond to the products of the ilvGMEDA and ilvDA genes. The transcripts were identified by the use of eight probes derived from segments of the ilvGMEDA cluster. In addition, we used two strains with deletions of ilvG or ilvDA and observed the expected decrease in transcript size in Northern blots. Primer extension with reverse transcriptase generated a 169-nucleotide product corresponding to a 5' end within the ilvED intercistronic region, 37 nucleotides from the AUG codon of the ilvD gene. This primer extension product presumably indicates the 5' end of the ilvDA transcript that we detected in Northern blots. The stability of the transcripts was monitored, and RNase E was found to play a major role in ilv transcript degradation. Transcript levels varied in response to growth in the presence of the end product amino acids and in response to the presence of the polar frameshift site in ilvG. Although there have been speculations about the identities and numbers of transcripts derived from the ilvGMEDA cluster on the basis of the identification of some of the sites of transcription initiation and termination, this is the first report of the use of Northern blots to determine the actual sizes and distribution of mRNAs present in vivo.
Mutations in a central highly conserved non-DNA-binding region of OmpR, an Escherichia coli transcriptional activator, influence its DNA-binding ability
Authors: Brissette R E , Tsung K , Inouye M
Published: November 2016

OmpR is a transcriptional activator for the expression of outer membrane porin genes ompF and ompC in Escherichia coli. Its C-terminal half has been identified as the DNA-binding domain (K. Tsung, R. Brissette, and M. Inouye, J. Biol. Chem. 264:10104-10109, 1989). Recent studies have indicated that the N-terminal non-DNA-binding domain of OmpR is involved in modulating OmpR function through interaction with the EnvZ protein, a kinase and phosphatase for OmpR. We isolated and characterized two mutations, G94D and E111K, in the N-terminal domain of OmpR and one mutation, R182C, in the DNA-binding domain of OmpR. All three mutations abolished the ability of OmpR to bind to the ompF and ompC promoters in vivo, thus giving an OmpF- OmpC- phenotype. The decreased DNA-binding ability of the mutant OmpRs was not due to diminished phosphorylation of their N termini, since all the mutant OmpRs were found to be normally phosphorylated by EnvZ in vitro. The mutant OmpRs produced from multicopy plasmids were also found to inhibit completely the production of OmpF and OmpC in wild-type cells, and the complete inhibition depended on the function of EnvZ which was produced in cis or in trans from plasmids. The relationship of the possible alterations in OmpR by the mutations with the observed diminished binding ability is discussed.
Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B
Authors: Létoffé S , Wandersman C
Published: November 2016

Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats. This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway. The role of these repeats in the secretion process is controversial. We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain. Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins. Secretion efficiency was also dependent on the size of the passenger polypeptide.
Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants
Authors: Nakayama K
Published: November 2016

A gene (sod) encoding superoxide dismutase (SOD) was cloned from Streptococcus mutans in Escherichia coli, and its nucleotide sequence was determined. The presumptive amino acid sequence of its product revealed that the SOD is basically of Mn type. Insertional inactivation of the sod gene resulted in the loss of SOD activity in crude extracts, indicating that the gene represents the only functional gene for SOD in S. mutans. Moreover, Southern blot analysis indicated that the S. mutans chromosome had no additional gene which was hybridizable with an oligonucleotide probe specific for an SOD motif. The SOD-deficient mutants were able to grow aerobically, albeit more slowly than the parent strains.
Localization of upstream sequence elements required for nitrate and anaerobic induction of fdn (formate dehydrogenase-N) operon expression in Escherichia coli K-12
Authors: Li J , Stewart V
Published: November 2016

Two transcriptional activators, the FNR and NARL proteins, are required for induction of the fdnGHI operon, encoding Escherichia coli formate dehydrogenase-N. The FNR protein is required for anaerobic expression, while the NARL protein mediates nitrate induction. We used primer extension to locate the transcription initiation site 29 nucleotides upstream of the fdnG translation initiation codon. Expression assays with single-copy phi (fdnG-lacZ) gene fusions containing various deletions in the fdn 5'-regulatory region delimited three distinct cis-acting elements. One site, which is located at approximately -110, was required for nitrate induction. Two other sites share sequence similarity with the FNR protein binding site core consensus. The first site, centered at -42.5, was required for anaerobic induction. We used site-specific mutagenesis to change this putative FNR protein binding site into the CRP protein binding site core consensus. This change caused the fdn operon to be expressed aerobically, subject to CRP protein control. On the other hand, converting this putative FNR protein binding site into the FNR protein binding site core consensus resulted in elevated anaerobic induction of the fdn operon and also caused weak aerobic expression. The other putative FNR protein binding site, centered at -97.5, was not involved in anaerobic induction. It might play a negative role in fdn operon expression during anaerobic growth in the absence of nitrate.
Lignin peroxidase gene family of Phanerochaete chrysosporium: complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome
Authors: Stewart P , Kersten P , Vanden Wymelenberg A , Gaskell J , Cullen D
Published: November 2016

Lignin peroxidases (LiP) of Phanerochaete chrysosporium are encoded by a family of six closely related genes. Five LiP genes have been localized to the same dimorphic chromosome. In this investigation, relative transcript levels of the LiP genes were determined. Transcripts of the LiPA, LiPB, and O282 genes were at similar levels in both carbon- and nitrogen-limited cultures. In contrast, transcription of the GLG5, V4, and GLG4 genes was dramatically altered by culture conditions. Under carbon-limited conditions, GLG4 transcripts were, by far, the most abundant. Southern blot analyses of clamped homogeneous field gels were used to map the GLG4 gene to a dimorphic chromosome separate from the other LiP genes.
Menaquinone (vitamin K2) biosynthesis: nucleotide sequence and expression of the menB gene from Escherichia coli
Authors: Sharma V , Suvarna K , Meganathan R , Hudspeth M E
Published: November 2016

In Escherichia coli, the biosynthesis of the electron carrier menaquinone (vitamin K2) involves at least seven identified enzymes. One of these, naphthoate synthase, forms the bicyclic ring system by catalyzing the conversion of o-succinylbenzoyl-coenzyme A to 1,4-dihydroxy-2-naphthoic acid. The gene for this enzyme has been previously identified as menB. By genetic and biochemical tests, a 1.349-kb DNA fragment from the E. coli men locus complements menB mutants. This fragment contains a single 285-codon open reading frame (ORF). Recombinant plasmids containing deletions of either the amino or the carboxy region of the ORF fail to complement the mutants. The ORF is preceded by consensus sequences for a ribosomal binding site and a sigma 70 promoter. menB transcription sufficient to complement the menB mutant in vivo and in vitro can be initiated from the identified putative promoter, and that in the constructs, menB expression, can be made independent of read-through transcription from the lac promoter. However, multicopy plasmids containing menB fail to generate the expected levels of enzymatic activity.