bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis
Authors: Molenaar D , Hagting A , Alkema H , Driessen A J , Konings W N
Published: November 2016

Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed.
Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator
Authors: Brumbley S M , Carney B F , Denny T P
Published: November 2016

Phenotype conversion (PC) in Pseudomonas solanacearum is the coordinated change in production of extracellular polysaccharide and a variety of extracellular proteins, some of which contribute to virulence. Although PC is normally spontaneous, it is mimicked by transposon inactivation of the phcA locus (S. M. Brumbley and T. P. Denny, J. Bacteriol. 172:5677-5685, 1990). The DNA sequence of a 1.8-kb region from strain AW1 that contains phcA revealed one open reading frame that should encode a polypeptide of 38.6 kDa. The PhcA protein produced in Escherichia coli by using a T7 RNA polymerase expression system was of the predicted size. The deduced amino acid sequence of PhcA is similar to that of some members of the LysR transcriptional activator gene family, especially in the amino terminus, where a putative helix-turn-helix DNA-binding motif was identified. An analogous allele (phcA1) was cloned from the spontaneous PC mutant strain AW1-PC and found to be nonfunctional in complementation studies. When phcA1 was expressed in E. coli, the PhcA1 protein was 35.5 kDa, 3 kDa smaller than PhcA. Sequence analysis of phcA1 and chimeric constructs of phcA and phcA1 confirmed that PhcA1 is truncated by a 2-bp insertion 147 nucleotides upstream of the carboxyl terminus of PhcA. Southern blot analysis of 10 additional independently isolated PC mutants of strain AW1 revealed that two strains have larger insertions (0.2 and 1.0 kb) within phcA. These results suggest that phcA encodes a DNA-binding protein that regulates the transcription of one or more of the genes involved in P. solanacearum virulence and that spontaneous PC can be attributed to one of several different insertions within this locus.
Cloning, primary structure, and regulation of the HIS7 gene encoding a bifunctional glutamine amidotransferase: cyclase from Saccharomyces cerevisiae
Authors: Kuenzler M , Balmelli T , Egli C M , Paravicini G , Braus G H
Published: November 2016

The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene. The two genes have the same orientation with a distance of only 416 bp between the two open reading frames. The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis. The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082. Two findings implicate a bifunctional nature of the HIS7 gene product. First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis. Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants. HIS7 gene expression is regulated by the general control system of amino acid biosynthesis. GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.
The Candida albicans plasma membrane and H(+)-ATPase during yeast growth and germ tube formation
Authors: Monk B C , Niimi M , Shepherd M G
Published: November 2016

PMA1 expression, plasma membrane H(+)-ATPase enzyme kinetics, and the distribution of the ATPase have been studied in carbon-starved Candida albicans induced with glucose for yeast growth at pH 4.5 and for germ tube formation at pH 6.7. PMA1 expression parallels expression of the constitutive ADE2 gene, increasing up to sixfold during yeast growth and twofold during germ tube formation. Starved cells contain about half the concentration of plasma membrane ATPase of growing cells. The amount of plasma membrane ATPase is normalized prior to either budding or germ tube emergence by the insertion of additional ATPase molecules, while ATPase antigen appears uniformly distributed over the entire plasma membrane surface during both growth phases. Glucose addition rapidly activates the ATPase twofold regardless of the pH of induction. The turnover of substrate molecules per second by the enzyme in membranes from budding cells quickly declines, but the enzyme from germ tube-forming cells maintains its turnover of substrate molecules per second and a higher affinity for Mg-ATP. The plasma membrane ATPase of C. albicans is therefore regulated at several levels; by glucose metabolism/starvation-related factors acting on gene expression, by signals generated through glucose metabolism/starvation which are thought to covalently modify the carboxyl-terminal domain of the enzyme, and possibly by additional signals which may be specific to germ tube formation. The extended period of intracellular alkalinization associated with germ tube formation may result from regulation of proton-pumping ATPase activity coupled with higher ratios of cell surface to effective cytosolic volume.
Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport
Authors: Hayman G T , Beck von Bodman S , Kim H , Jiang P , Farrand S K
Published: November 2016

The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy. acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to be located in the periplasm. The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis.
The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein
Authors: Mengaud J M , Horwitz M A
Published: November 2016

Legionella pneumophila has high iron requirements, and its intracellular growth in human monocytes is dependent on the availability of intracellular iron. To learn more about iron metabolism in L. pneumophila, we have undertaken an analysis of the iron proteins of the bacterium. We first developed an assay to identify proteins by 59Fe labelling and nondenaturing polyacrylamide gel electrophoresis. The assay revealed seven iron proteins (IPs) with apparent molecular weights of 500, 450, 250, 210, 150, 130, and 85. IP150 comigrates with superoxide dismutase activity and is probably the Fe-superoxide dismutase of L. pneumophila. IP210 is the major iron-containing protein (MICP). To identify and characterize MICP, we purified the protein and cloned and sequenced its gene. MICP is a monomeric protein containing 891 amino acids, and it has a calculated molecular mass of 98,147 Da. Analysis of the sequence revealed that MICP has two interesting homologies. First, MICP is highly homologous with the human iron-responsive element-binding protein, consistent with the hypothesis that this critical iron-regulatory molecule of humans has a prokaryotic ancestor. Second, MICP is highly homologous with the Escherichia coli aconitase and to a lesser extent with porcine heart mitochondrial aconitase. Consistent with this, we found that MICP exhibits aconitase activity. In contrast to other aconitases, MICP has a single amino acid change of a potentially deleterious type at a site thought to be critical for substrate binding and enzymatic activity. However, the specific activity of MICP is roughly comparable to that of other aconitases, suggesting that the mutation has at most a mild effect on the aconitase activity of MICP. The abundance of MICP in L. pneumophila suggests either that L. pneumophila requires high aconitase and perhaps tricarboxylic acid cycle activity or that the bacterium requires large amounts of this protein to serve an additional role in bacterial physiology. A need for large amounts of MICP, which contains four Fe atoms per molecule when fully loaded, could at least partly explain L. pneumophila's high metabolic requirement for iron.
Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis
Authors: Kemper M A , Urrutia M M , Beveridge T J , Koch A L , Doyle R J
Published: November 2016

Bacterial metabolism excretes protons during normal metabolic processes. The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents. Calculations by Koch (J. Theor. Biol. 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH. That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments. When cultures of B. subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the cells bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO2(2+) ions and were readily agglutinated by cationized ferritin. In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (CrO4(2-)). Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth. The composite interpretation of the foregoing observations suggests that the wall is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions. This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium.
Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative
Authors: Tascon R I , Rodriguez-Ferri E F , Gutierrez-Martin C B , Rodriguez-Barbosa I , Berche P , Vazquez-Boland J A
Published: November 2016

A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed.
Limited differential mRNA inactivation in the atp (unc) operon of Escherichia coli
Authors: Lagoni O R , von Meyenburg K , Michelsen O
Published: November 2016

Individual subunits of ATP synthase, encoded by the eight genes of the atp operon (atpA through atpH), have been found to be synthesized at a 10-fold range in molar amounts (D.L. Foster and R.H. Fillingame, J. Biol. Chem. 257:2009-2015, 1982; K. von Meyenburg, B.B. Jorgensen, J. Nielsen, F.G. Hansen, and O. Michelsen. Tokai J. Exp. Clin. Med. 7:23-31, 1982). We have determined the functional half-lives at 30 degrees C of mRNAs transcribed from these genes either during constitutive expression in a partial diploid strain or after induced expression from a plasmid. Accurate decay kinetics of the relative mRNA levels were determined by monitoring the rates of synthesis of the individual ATP synthase subunits by radioactive pulse labeling at different times after blocking transcription initiation with rifampin. The mRNA transcribed from the atp operon was found to be inactivated about twice as fast as the bulk mRNA in E. coli. Exceptions are the mRNA from the promoter-proximal atpB gene, which was inactivated about three times as fast as the bulk mRNA, and atpC mRNA, the inactivation rate of which was comparable to that of the bulk mRNA. These moderate differences in the kinetics of functional decay explain only a minor part of the differences in expression levels of the atp genes. We conclude, therefore, that the individual atp mRNAs must be translated with widely different efficiencies. The present analysis further revealed that mRNA degradation is sensitive to heat shock; i.e., after incubation at 39 degrees C for 5 min followed by a shift back to 30 degrees C, the decay rate of the bulk mRNA was decreased by 30%.
Regulation of the pcaIJ genes for aromatic acid degradation in Pseudomonas putida
Authors: Parales R E , Harwood C S
Published: November 2016

Six of the genes encoding enzymes of the beta-ketoadipate pathway for benzoate and 4-hydroxybenzoate degradation in Pseudomonas putida are organized into at least three separate transcriptional units. As an initial step to defining this pca regulon at the molecular level, lacZ fusions were made with the pcaI and pcaJ genes, which encode the two subunits of beta-ketoadipate:succinyl-coenzyme A transferase, the enzyme catalyzing the next-to-last step in the beta-ketoadipate pathway. Fusion analyses showed that pcaI and pcaJ constitute an operon which requires beta-ketoadipate or its nonmetabolizable analog, adipate, as well as the pcaR regulatory gene for induction. The pcaIJ promoter is likely to be a sigma 70-type promoter; it has a sigma 70-type consensus sequence and did not require the alternative sigma factor, RpoN, for induction. Deletion analysis of the promoter region of a pcaI-lacZ transcriptional fusion indicated that no specific DNA sequences upstream of the -35 region were required for full induction. This implies that the binding site for the activator protein, PcaR, is unusually close to the transcriptional start site of pcaIJ.
The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY
Authors: Darzins A
Published: November 2016

The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili.
Methyl viologen hydrogenase II, a new member of the hydrogenase family from Methanobacterium thermoautotrophicum delta H
Authors: Woo G J , Wasserfallen A , Wolfe R S
Published: November 2016

Two methyl viologen hydrogenase (MVH) enzymes from Methanobacterium thermoautotrophicum delta H have been separated (resolution, Rs at 1.0) on a Mono Q column after chromatography on DEAE-Sephacel and Superose 6 Prep Grade. The newly discovered MVH (MVH II) was eluted at 0.5 M NaCl with a linear gradient of 0.45 to 0.65 M NaCl (100 ml). The previously described MVH (MVH I) eluted in a NaCl gradient at 0.56 M. The specific activities of MVH I and MVH II were 184.8 and 61.3 U/mg of protein, respectively, when enzyme activity was compared at pH 7.5, the optimal pH for MVH II. Gel electrophoresis in nondenaturing systems indicated that MVH I and MVH II had a similar molecular mass of 145 kDa. Denatured MVH II showed four protein bands (alpha, 50 kDa; beta, 44 kDa; gamma, 36 kDa; delta, 15 kDa), similar to MVH I. The N-terminal amino acid sequences of the alpha, gamma, and delta subunits of MVH II were identical with the sequences of the equivalent subunits of MVH I. However, the N-terminal amino acid sequence of the beta subunit of MVH II was totally different from the sequence of the beta subunit of MVH I. Both MVH I and MVH II had the same optimal temperature of 60 degrees C for maximum activity. The pH optima of MVH I and MVH II were 9.0 and 7.5, respectively. Most of the divalent metal ions tested significantly inhibited MVH I activity, but MVH II activity was only partially inhibited by some divalent cations. Both hydrogenases were shown to be stable for over 8 days at --20 degrees C under anaerobic conditions. When exposed to air, 90% of MVH I activity was lost within 2 min; however, MVH II lost only 50% of its activity in 3 h.
Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide
Authors: Pazzani C , Rosenow C , Boulnois G J , Bronner D , Jann K , Roberts I S
Published: November 2016

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters.
Analysis of a region from the bacteriophage resistance plasmid pCI528 involved in its conjugative mobilization between Lactococcus strains
Authors: Lucey M , Daly C , Fitzgerald G
Published: November 2016

A 10-kb HindIII fragment of pCI528 cloned into the nonconjugative shuttle vector pCI3340 could be transferred by conjugative mobilization from Lactococcus lactis subsp. lactis MG1363, whereas other HindIII fragments of pCI528 or the vector alone were nonmobilizable. Subcloning of this 10-kb region identified a 4.4-kb BglII-EcoRI fragment which contained all the DNA essential for transfer. Sequence analysis of a 2-kb region within this 4.4 kb-segment revealed a region rich in inverted repeats and two potential overlapping open reading frames, one of which demonstrated homology to mobilization proteins of two nonconjugative staphylococcal plasmids.
Three distinct secreted aspartyl proteinases in Candida albicans
Authors: White T C , Miyasaki S H , Agabian N
Published: November 2016

The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct proteinase isoenzymes, two of which correspond to the recently cloned SAP1 and SAP2 genes (previously referred to as CAP, PEP, or PRA). A genomic library was screened under low-stringency hybridization conditions with a polymerase chain reaction fragment from SAP1. In addition to clones of SAP1 and SAP2, a clone containing SAP3, a novel third secreted proteinase gene, was identified and sequenced. The three aspartyl proteinase isoenzymes differ in primary sequence and pI, suggesting that they may play different roles in virulence and pathogenesis. All three of these proteinases are expressed in the same strain. However, the pattern of proteinase expression is correlated with the switch phenotype of the cell. Opaque cells of strain WO-1 express Sap1 and Sap3, while white cells of the same strain express Sap2. The differential expression of three Sap proteinases may contribute to virulence in C. albicans.
Molecular characterization of the Enterobacter aerogenes tonB gene: identification of a novel type of tonB box suppressor mutant
Authors: Bruske A K , Heller K J
Published: November 2016

The tonB gene of Enterobacter aerogenes was cloned, sequenced, and expressed in Escherichia coli. It complemented an E. coli tonB mutant as efficiently as E. coli tonB, except for colicin B and D sensitivities. However, colicin B and D sensitivities were complemented by a derivative in which the aspartate at position 165 was replaced by a glutamine (TonBD-165-->Q) by site-directed mutagenesis. In E. coli, the corresponding amino acid is a glutamine (Q-160) which is known to be altered in most mutants showing suppression of the btuB451 mutation. Fourteen independent btuB451 suppressor mutations in E. aerogenes tonB which all had suffered the same point mutation resulting in a change from glycine to valine at position 239 (G-239-->V) of the C-terminal end of the protein were isolated. The mutation was located within a region which is nonessential for function of E. aerogenes TonB as well as E. coli TonB. A constructed double mutation, expressing a D-165-->Q/G-239-->V derivative, no longer acted as a btuB451 suppressor. However, it restored colicin B and D sensitivities even more efficiently than the D-165-->Q derivative. Corresponding mutations constructed in E. coli tonB, giving rise to Q-160-->D, G-234-->V, and Q-160-->D/G-234-->V derivatives, showed phenotypes comparable to the E. aerogenes mutations. We take this as evidence that at least a functional interaction between the D-165 (Q-160 in E. coli) and the G-239 (G-234 in E. coli) region is necessary for TonB function. The implications of this interaction for functional instability of TonB are discussed.
vsrB, a regulator of virulence genes of Pseudomonas solanacearum, is homologous to sensors of the two-component regulator family
Authors: Huang J , Denny T P , Schell M A
Published: November 2016

Pseudomonas solanacearum, an important wilt pathogen of many plants, produces several extracellular proteins (EXPs) and extracellular polysaccharides (EPSs) that contribute to its virulence. Using TnphoA mutagenesis, we discovered a new gene, vsrB, that when inactivated causes a major reduction in the virulence and production of an EPS. Analysis of eps::lacZ reporters showed that vsrB is required for maximal expression (transcription) of eps, whose products are required for production of EPS I, a major virulence determinant. Analysis of EXPs in culture supernatants revealed that inactivation of vsrB also causes reduced production of two major EXPs, with molecular masses of 28 and 97 kDa, and a simultaneous 15-fold increase in levels of another EXP, PglA endopolygalacturonase. The vsrB gene was cloned from a P. solanacearum genomic library by complementation of the nonmucoid phenotype of the vsrB::TnphoA mutant and then subcloned on a 2.4-kb DNA fragment. TnphoA fusion analysis and subcellular localization of the vsrB gene product in Escherichia coli maxicells suggest that it is a ca. 60-kDa transmembrane protein. The nucleotide sequence of the 2.4-kb DNA fragment was determined, and a 638-amino-acid open reading frame was found for VsrB. A search of the GenBank data base found that the central part of VsrB has homology with the histidine kinase domain of sensors in the two-component regulator family, while the C terminus has homology with the phosphate receiver domain of response regulators in the same family. Genetic analysis suggests that the receiver domain is not required for vsrB function.
Multiple factors underlying the maximum motility of Escherichia coli as cultures enter post-exponential growth
Authors: Amsler C D , Cho M , Matsumura P
Published: November 2016

Motility and chemotaxis allow cells to move away from stressful microenvironments. Motility of Escherichia coli in batch cultures, as measured by cell swimming speed, was low in early-exponential-phase cells, peaked as the cells entered post-exponential phase, and declined into early stationary phase. Transcription from the flhB operon and synthesis of flagellin protein similarly peaked in late exponential and early post-exponential phases, respectively. The increase in swimming speed between early-exponential and post-exponential phases was correlated with twofold increases in both flagellar length and flagellar density per cell volume. This increased investment in flagella probably reflects the increased adaptive value of motility in less favorable environments. The decrease in speed between post-exponential and stationary phases was correlated with a threefold decrease in torque produced by the flagellar motors and presumably reflects decreased proton motive force available to stationary-phase cells.
Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis
Authors: Thiel T
Published: November 2016

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.
Induction of EcoRII methyltransferase: evidence for autogenous control
Authors: Friedman S , Som S
Published: November 2016

The cytosine analog 5-azacytidine kills Escherichia coli cells that carry plasmids expressing EcoRII DNA (cytosine 5)methyltransferase under control of its own promoter. We previously showed that this enzyme binds tightly to azacytidine-containing DNA in vitro and proposed that such binding is lethal in vivo. In support of this proposal, we now show that the enzyme sediments with the nucleoid of azacytidine-treated cells. Azacytidine treatment led to an increase in the amount of enzyme, and this increase required sequences in the ecoRIIM promoter region. Enzyme inducibility correlated with drug sensitivity: plasmids carrying the methyltransferase gene but lacking the wild-type promoter did not confer sensitivity. These results suggested that the ecoRIIM gene was under autogenous control. Transcriptional ecoRIIM'-lacZ fusions in E. coli were, therefore, constructed. They showed that expression from the ecoRIIM promoter was inhibited when EcoRII DNA (cytosine-5)methyltransferase was introduced into the cell in trans and inhibition was reversed by treating the cells with azacytidine. These results provide evidence that the expression of the ecoRIIM gene is under autogenous regulation and that cell death induced by azacytidine is due, in part, to the disruption of autoregulation.
A growth rate-limiting process in the last growth phase of the yeast life cycle involves RPB4, a subunit of RNA polymerase II
Authors: Choder M
Published: November 2016

Saccharomyces cerevisiae cells, grown on a fermentable carbon source, display two growth phases before they enter the stationary phase: a rapid phase (log phase) followed by a slow phase. It was previously shown that a subunit of the yeast RNA polymerase II, RPB4, positively affects the activity of the enzyme in post-log phases but has little or no effect on its activity in log phase. Here, I show that RPB4 level limits the growth rate during the slow growth phase. Thus, a small increase in RPB4 protein level, in cells carrying multiple copies of the RPB4 gene, results in an almost twofold increase in the growth rate during this phase. Furthermore, RPB4 expression is differentially regulated in the two growth phases. During the slow growth phase, a posttranscriptional process which controls the RPB4 level and thus can control growth rate becomes active. These results reveal a complex growth control mechanism, in which the transcriptional apparatus is probably a limiting element, operating in the last stages of the yeast growth.
Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600
Authors: Powlowski J , Sahlman L , Shingler V
Published: November 2016

The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp. strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) [acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10]. A procedure for purifying these two enzyme activities to homogeneity is reported here. The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa. Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes. The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex. In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol. 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures. The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.
Organization and regulation of the mannopine cyclase-associated opine catabolism genes in Agrobacterium tumefaciens 15955
Authors: Hong S B , Dessaux Y , Chilton W S , Farrand S K
Published: November 2016

We have isolated and characterized Tn3HoHo1- and Tn5-induced mutants of a cosmid clone, pYDH208, which encodes the mannopine (MOP) cyclase-associated catabolism of MOP and agropine (AGR). Characterization of the transposon-induced lacZ fusion mutants by beta-galactosidase activity and mannityl opine utilization patterns identified at least 6 genetic units associated with the catabolism of these opines. Functions for the catabolism of MOP and mannopinic acid are encoded by a 16.4-kb region, whereas those for AGR are encoded by a 9.4-kb region located within the MOP catabolic locus. The induction pattern of catabolism shown by transposon insertion derivatives suggests that the catabolism of MOP, AGR, and mannopinic acid encoded by pYDH208 is regulated by at least two independent control elements. Kinetic uptake assays indicate that the clone encodes two transport systems for MOP and AGR, one constitutive and slow and the other inducible and rapid. Analysis of beta-galactosidase activities from lacZ reporter gene fusions indicated that expression of mannityl opine catabolic genes is not strongly repressed by sugars but is repressed by succinate when ammonium is the nitrogen source. The repression exerted by succinate was relieved when MOP was supplied as the sole source of nitrogen. This suggests that genes for opine catabolism encoded by pYDH208 are regulated, in part, by nitrogen availability.
Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816
Authors: van Rhijn P J , Feys B , Verreth C , Vanderleyden J
Published: November 2016

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.
Identification and characterization of RP1 Tra1 cistrons involved in pilus function and plasmid mobilization
Authors: Fong S T , Stanisich V A
Published: November 2016

Transfer-defective mutants of the Tra1 region of RP1 were isolated. Complementation studies involving stable heterozygotes combined with the mapping of Tn5 insertion mutations revealed two pilus cistrons, pilA and pilB, at positions 46.9 to 48.2 kb and 46.0 to 46.4 kb, respectively. All pilB mutants were Dps- (i.e., resistant to donor-specific phages PR4 and PRR1), whereas pilA mutants were Dps- (promoter-proximal mutations), Dps+/- (sensitive only to PR4 [more centrally located mutations]), or Dps+ (sensitive to both phages [promoter-distal mutations]). The correlation between the site mutated and the Dps phenotype, together with the finding that certain Dps+ pilA mutants continued to mobilize nonconjugative plasmids, suggested that pilA is bifunctional, contributing both to pilus function (at the promoter-proximal end) and to RP1 mobilization. It was also shown that the 43.5- to 49.5-kb region that includes pilA and pilB encodes all of the Tra1 pilus functions required for propagation of donor-specific phages and hence, probably, for pili that are active in conjugation. Finally, three cistrons that specifically affect RP1 mobilization were identified. Two of these, mobA and mobB, occur immediately anticlockwise to oriT and probably correspond to the traJ and traI genes characterized by other workers. The third cistron, mobC, occurs clockwise to oriT and may be a new mobilization gene, since its function can be substituted by IncP beta plasmids, a feature different from that of the traK mobilization gene which occurs in the same region but is RP1 specific. None of the mob cistrons was required for mobilization of nonconjugative plasmids, except for mobB, which was required by pVS99.
The Salmonella typhimurium nadC gene: sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyltransferase
Authors: Hughes K T , Dessen A , Gray J P , Grubmeyer C
Published: November 2016

The Salmonella typhimurium nadC gene and its product, quinolinic acid phosphoribosyltransferase (QAPRTase), were characterized at the molecular and biochemical levels. Fusions of Mud-lac elements isolated in the nadC gene were converted to Mud-P22 insertions. Starting with six original Mud-lac fusions, the entire sequence of the nadC gene was readily obtained. The sequence shows a long open reading frame with two potential initiator methionines, one of which is preceded by the Shine-Dalgarno sequence GGAG-7-nucleotide-ATG. The protein predicted from this second open reading frame is 297 residues in length. The nadC gene was subcloned into a T7-based expression system, allowing for facile purification of the QAPRTase (EC 2.4.2.19) protein to homogeneity. Upon gel filtration, the protein gave an M(r) of 72,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a subunit M(r) of 35,000. Automated Edman degradation of several tryptic peptides confirmed the amino acid sequence predicted from the DNA sequence. Chromatography of the apparently homogeneous enzyme on reverse-phase high-performance liquid chromatography resolved two protein species. One of these species failed to give an amino-terminal sequence, while the other yielded the amino-terminal sequence predicted by the second open reading frame and lacked the initiator methionine. The mass of the mature protein, predicted from its DNA sequence, was 32,428 Da. Electrospray mass spectrometry gave masses of 32,501 and 32,581 Da for the two peptides. Steady-state kinetics on the purified QAPRTase indicated Km values of 32 microM for 5-phosphoribosyl-1-pyrophosphate and 20 microM for quinolinate. Vmax was 0.9 U/mg, similar to values reported for this enzyme by other sources.
Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species
Authors: Kaltenboeck B , Kousoulas K G , Storz J
Published: November 2016

DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies.
Repressor binding to a regulatory site in the DNA coding sequence is sufficient to confer transcriptional regulation of the vir-repressed genes (vrg genes) in Bordetella pertussis
Authors: Beattie D T , Mahan M J , Mekalanos J J
Published: November 2016

Five TnphoA fusions to vir-repressed genes (vrg genes) have been identified in the respiratory pathogen Bordetella pertussis. A comparison of vrg DNA sequences suggests a consensus DNA element within the coding regions of four of five vrg genes. To determine the role of this DNA sequence in vrg regulation, a nucleotide substitution mutation in the conserved region of vrg-6 was isolated. This mutant showed constitutively high levels of expression in the absence of antigenic modulators, MgSO4 and nicotinic acid, suggesting that this DNA element may be a control site for vrg repression. Moreover, Northern (RNA) analysis and transcriptional fusion analysis suggest that vrg genes are regulated at the transcriptional level. To determine whether sequences in the coding region were sufficient to respond to antigenic modulation, a vrg-6::TnphoA promoter deletion plasmid that contained a heterologous promoter driving the expression of vrg-6 coding sequences from the vrg-6 translation start site to the TnphoA fusion junction was constructed. This heterologous construct responded to modulators in a vir-dependent fashion, indicating that sequences upstream of the coding sequence are not required for antigenic modulation. Southwestern (DNA-protein) analysis and mutational studies suggest that the vrg consensus DNA sequence is specifically recognized by a 34-kDa vir-activated gene (vag) product, whose binding results in down-regulation of vrg transcript levels. We conclude, at least for the vrg::TnphoA fusion strains, that a site on the DNA that corresponds to a consensus sequence located in the vrg coding region is sufficient to confer the transcriptional regulation (repression) of vrg genes when B. pertussis strains are grown under nonmodulating conditions.
Sporulation gene spoIIB from Bacillus subtilis
Authors: Margolis P S , Driks A , Losick R
Published: November 2016

We have cloned and characterized the sporulation gene spoIIB from Bacillus subtilis. In extension of previous nucleotide sequence analysis, our results show that the order of genes in the vicinity of spoIIB is valS folC comC spoIIB orfA orfB mreB mreC mreD minC minD spoIVFA spoIVFB L20 orfX L24 spoOB obg pheB pheA. All 20 genes have the same orientation; the direction of transcription is from valS to pheA. We show that spoIIB is a 332-codon-long open reading frame whose transcription is under sporulation control. The deduced amino acid sequence of the spoIIB gene product, a 36-kDa polypeptide, is highly charged and contains a stretch of uncharged amino acids that could correspond to a transmembrane segment. Surprisingly, mutations in spoIIB, including an in vitro-constructed null mutation, cause only a mild impairment of spore formation in certain otherwise wild-type bacteria. However, when combined with mutations in another sporulation gene, spoVG, mutations in spoIIB cause a severe block in spore formation at the stage (stage II) of septum formation. (As with spoIIB mutations, mutations in spoVG cause little impairment in sporulation on their own.) The nature of the spoIIB spoVG mutant phenotype is discussed in terms of the events involved in the maturation of the sporulation septum and in the activation of sporulation transcription factors sigma F and sigma E.