bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by cyclic AMP-dependent protein kinase
Authors: Kinney A J , Bae-Lee M , Panghaal S S , Kelley M J , Gaynor P M , Carman G M
Published: November 2016

The addition of cyclic AMP (cAMP) to Saccharomyces cerevisiae cyr1 mutant cells resulted in an increase in the rate of phosphatidylinositol synthesis at the expense of phosphatidylserine synthesis. The decrease in phosphatidylserine synthesis correlated with the down regulation of phosphatidylserine synthase activity by cAMP-dependent protein kinase phosphorylation. The increase in phosphatidylinositol synthesis was not due to the regulation of phosphatidylinositol synthase by cAMP-dependent protein kinase.
Identification of BlaR, the signal transducer for beta-lactamase production in Bacillus licheniformis, as a penicillin-binding protein with strong homology to the OXA-2 beta-lactamase (class D) of Salmonella typhimurium
Authors: Zhu Y F , Curran I H , Joris B , Ghuysen J M , Lampen J O
Published: November 2016

The blaR gene of Bacillus licheniformis encodes the signal transducer for induction of the class A beta-lactamase. The protein product, BlaR, has a hydrophilic carboxy region that binds beta-lactams and shows high sequence homology to the class D beta-lactamases, particularly the OXA-2 beta-lactamase of Salmonella typhimurium. The BlaR-beta-lactam complex is stable and may provide the continuing stimulus needed for the prolonged production of the enzyme.
The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation
Authors: Jin S , Roitsch T , Ankenbauer R G , Gordon M P , Nester E W
Published: November 2016

The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. We overproduced truncated VirA proteins in Escherichia coli by deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [gamma-32P]ATP but not with [gamma-32P]GTP or [alpha-32P]ATP, which suggests an ATP gamma-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens.
The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes
Authors: Jin S G , Roitsch T , Christie P J , Nester E W
Published: November 2016

Virulence genes of Agrobacterium tumefaciens are induced in parallel in the presence of plant phenolic compounds such as acetosyringone and the two regulatory vir genes virA and virG. In this study we identified a cis-acting regulatory sequence in the 5'-noncoding region of the virE operon that is essential for this activation. To do this, we constructed a series of deletion mutants by using exonuclease Bal 31. Western blot (immunoblot) analysis showed that the 70 base pairs upstream of the transcriptional start site were sufficient for full virE gene induction. A conserved dodecadeoxynucleotide sequence (vir box), which was previously identified in the nontranscribed sequences of all vir genes, was located at 5' end of the minimum required promoter sequence. Deletion of this vir box only completely abolished induction of the virE gene. This demonstrates that the vir box functions as an upstream regulatory sequence. To study the role of the VirG protein in the activation process, we overproduced the native-sized VirG protein in Escherichia coli by fusing the lacZ' start codon ATG with the second virG codon AAA using site-directed mutagenesis. The VirG protein was purified and renatured from E. coli and was shown to bind to a specific sequence in two vir gene promoters. Footprinting analysis of the virE and virB promoters identified the 12-base-pair vir box as the VirG-binding core sequence.
Characterization of transmembrane movement of glucose and glucose analogs in Streptococcus mutants Ingbritt
Authors: Dashper S G , Reynolds E C
Published: November 2016

The transmembrane movement of radiolabeled, nonmetabolizable glucose analogs in Streptococcus mutants Ingbritt was studied under conditions of differing transmembrane electrochemical potentials (delta psi) and pH gradients (delta pH). The delta pH and delta psi were determined from the transmembrane equilibration of radiolabeled benzoate and tetraphenylphosphonium ions, respectively. Growth conditions of S. mutants Ingbritt were chosen so that the cells had a low apparent phosphoenolpyruvate (PEP)-dependent glucose:phosphotransferase activity. Cells energized under different conditions produced transmembrane proton potentials ranging from -49 to -103 mV but did not accumulate 6-deoxyglucose intracellularly. An artificial transmembrane proton potential was generated in deenergized cells by creating a delta psi with a valinomycin-induced K+ diffusion potential and a delta pH by rapid acidification of the medium. Artificial transmembrane proton potentials up to -83 mV, although producing proton influx, could not accumulate 6-deoxyglucose in deenergized cells or 2-deoxyglucose or thiomethylgalactoside in deenergized, PEP-depleted cells. The transmembrane diffusion of glucose in PEP-depleted, KF-treated cells did not exhibit saturation kinetics or competitive inhibition by 6-deoxyglucose or 2-deoxyglucose, indicating that diffusion was not facilitated by a membrane carrier. As proton-linked membrane carriers have been shown to facilitate diffusion in the absence of a transmembrane proton potential, the results therefore are not consistent with a proton-linked glucose carrier in S. mutans Ingbritt. This together with the lack of proton-linked transport of the glucose analogs suggests that glucose transmembrane movement in S. mutans Ingbritt is not linked to the transmembrane proton potential.
Purification and properties of 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri
Authors: te Brömmelstroet B W , Hensgens C M , Geerts W J , Keltjens J T , van der Drift C , Vogels G D
Published: November 2016

The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability. Like the cyclohydrolase purified from Methanobacterium thermoautotrophicum (A. A. Dimarco, M. I. Donnelly, and R. S. Wolfe, J. Bacteriol. 168:1372-1377, 1986), the protein showed an apparent Mr of 82,000, and it is composed of two identical subunits as was concluded from nondenaturating and denaturating polyacrylamide gel electrophoresis. The enzymes from M. thermoautotrophicum and M. barkeri markedly differ with respect to the hydrolysis product of 5,10-methenyltetrahydromethanopterin: 5-formyl- and 10-formyltetrahydromethanopterin, respectively. The apparent Km for 5,10-methenyltetrahydromethanopterin was 0.57 mM at 37 degrees C and pH 7.8.
Genetic analysis of an invasion region by use of a Tn3-lac transposon and identification of a second positive regulator gene, invE, for cell invasion of Shigella sonnei: significant homology of invE with ParB of plasmid P1
Authors: Watanabe H , Arakawa E , Ito K , Kato J , Nakamura A
Published: November 2016

We have previously cloned two distinct regions of the Shigella sonnei form I plasmid pSS120, a 37-kilobase-pair DNA region and a virF region, which were found to be essential for cell invasion in Escherichia coli K-12 (J. Kato, K. Ito, A. Nakamura, and H. Watanabe, Infect. Immun. 57:1391-1398, 1989). The 37-kilobase-pair DNA region was randomly inserted by use of transposon Tn3-lac. At least eight genes were found to be located within the region, as determined by analysis of Tn3-lac-generated lac fusions. Expression of six genes, ipaB, ipaC, invE, invG, invJ, and invK, was apparently regulated by the positive regulator virF. IpaB and IpaC proteins could not found in invE mutants even if the virF gene was present. This observation suggested that the invE region encoded a positive regulator different from the virF gene. The functional relationship between the invE and virF genes was then examined. Translational fusions ipaB::Tn3-lac and invJ::Tn3-lac were used as indicators for gene expression, and the following results were obtained. Full expression of the ipaB and invJ genes required the presence of both the invE and virF regions. virF positively regulated the expression of invE at the transcriptional level. An increase in the copy number of invE enhanced the expression of ipaB and invJ in the absence of virF. These findings strongly indicate that the invE gene product, whose expression is regulated by virF, acts positively on the invasion-associated genes. InvE is a 35,407-dalton protein and has significant homologies with ParB of plasmid P1 and SopB of plasmid F, which are DNA-binding proteins involved in plasmid partitioning.
Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis
Authors: Jackson M P , Wadolkowski E A , Weinstein D L , Holmes R K , O'Brien A D
Published: November 2016

The B subunit of Shiga toxin and the Shiga-like toxins (SLTs) mediates receptor binding, cytotoxic specificity, and extracellular localization of the holotoxin. While the functional receptor for Shiga toxin, SLT type I (SLT-I), and SLT-II is the glycolipid designated Gb3, SLT-II variant (SLT-IIv) may use a different glycolipid receptor. To identify the domains responsible for receptor binding, localization in Escherichia coli, and recognition by neutralizing monoclonal antibodies, oligonucleotide-directed site-specific mutagenesis was used to alter amino acid residues in the B subunits of Shiga toxin and SLT-IIv. Mutagenesis of a well-conserved hydrophilic region near the amino terminus of the Shiga toxin B subunit rendered the molecule nontoxic but did not affect immunoreactivity or holotoxin assembly. In addition, elimination of one cysteine residue, as well as truncation of the B polypeptide by 5 amino acids, caused a total loss of activity. Changing a glutamate to a glutamine at the carboxyl terminus of the Shiga toxin B subunit resulted in the loss of receptor binding and immunoreactivity. However, the corresponding mutation in the SLT-IIv B subunit (glutamine to glutamate) did not reduce the levels of cytotoxicity but did affect extracellular localization of the holotoxin in E. coli.
RcsB and RcsC: a two-component regulator of capsule synthesis in Escherichia coli
Authors: Stout V , Gottesman S
Published: November 2016

Colanic acid capsule synthesis in Escherichia coli K-12 is regulated by RcsB and RcsC. The amino acid sequences of these two proteins, deduced from the nucleotide sequence reported here, demonstrate their homology to environmentally responsive two-component regulators that have been reported in both gram-positive and gram-negative bacteria. In our model, RcsC acts as the sensor and RcsB acts as the receiver or effector to stimulate capsule synthesis from cps genes. In addition, RcsC shows limited homology to the other effectors in its C terminus. Fusions of rcsC to phoA that resulted in PhoA+ strains demonstrated that RcsC is a transmembrane protein with a periplasmic N-terminal domain and cytoplasmic C-terminal domain. Additional control of this regulatory network is provided by the dependence on the alternate sigma factor, RpoN, for the synthesis of RcsB. The rcsB and rcsC genes, which are oriented convergently with their stop codons 196 base pairs apart, are separated by a long direct repeat including two repetitive extragenic palindromic sequences.
Effects of mutations in the repA gene of plasmid Rts1 on plasmid replication and autorepressor function
Authors: Terawaki Y , Nozue H , Zeng H , Hayashi T , Kamio Y , Itoh Y
Published: November 2016

We constructed a system in which wild-type RepA or RepAcop1 protein was supplied in trans in various amounts to coexisting mini-Rts1 plasmids by clones of the repA or repAcop1 gene under the control of the native promoter with or without its operator sequence. RepAcop1 protein which contains a single amino acid substitution (Arg-142 to Lys) within its 288 amino acids could initiate the replication of the mini-Rts1 plasmid efficiently at both 37 and 42 degrees C even if it was supplied in excess. In contrast, excess wild-type RepA inhibited plasmid replication at 37 degrees C but supported replication at 42 degrees C. Therefore, it appears that the initiator activity of RepA is not related to the incompatibility phenotype associated with an excess of RepA protein. An immunoblot analysis revealed that neither RepA nor RepAcop1 synthesis was temperature sensitive and that both were autogenously regulated to a similar extent because of the presence of an operator located immediately upstream of the promoter. Two mutant RepA proteins, each of which contains a 4-amino-acid insertion in the middle of the protein, maintained the autorepressor and incompatibility activities but lost the ori(Rts1)-activating function.
Genetic map of the Bacillus stearothermophilus NUB36 chromosome
Authors: Vallier H , Welker N E
Published: November 2016

A circular genetic map of Bacillus stearothermophilus NUB36 was constructed by transduction with bacteriophage TP-42C and protoplast fusion. Sixty-four genes were tentatively assigned a cognate Bacillus subtilis gene based on growth response to intermediates or end products of metabolism, cross-feeding, accumulation of intermediates, or their relative order in a linkage group. Although the relative position of many genes on the Bacillus stearothermophilus and Bacillus subtilis genetic map appears to be similar, some differences were detected. The tentative order of the genes in the Bacillus stearothermophilus aro region is aspB-aroBAFEC-tyrA-hisH-(trp), whereas it is aspB-aroE-tyrA-hisH-(trp)-aroHBF in Bacillus subtilis. The aroA, aroC, and aroG genes in Bacillus subtilis are located in another region. The tentative order of genes in the trp operon of Bacillus stearothermophilus is trpFCDABE, whereas it is trpABFCDE in Bacillus subtilis.
Structure of macroamphiphiles from several Bifidobacterium strains
Authors: Iwasaki H , Araki Y , Ito E , Nagaoka M , Yokokura T
Published: November 2016

Lipoteichoic acid-like substances, macroamphiphiles, were isolated from cell homogenates of Bifidobacterium bifidum YIT 4007 and YIT 4013, Bifidobacterium breve YIT 4010 and YIT 4014, and Bifidobacterium longum YIT 4021 by phenol extraction followed by nuclease digestion, gel chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography. The macroamphiphile preparations from these five strains contained D-glucose, D-galactose, glycerol, phosphorus, L-alanine, and fatty acids in molar ratios of 1.00, 1.57 to 1.95, 1.02 to 1.99, 0.97 to 1.72, 0.15 to 0.46, and 0.16 to 0.43. Data from structural analyses including methylation, 1H nuclear magnetic resonance measurement, alkaline hydrolysis, mild acid hydrolysis, and hydrogen fluoride treatment led to the most likely common structure for the macroamphiphiles of the examined strains, (formula; see text) where Gro-P is glycerophosphate, m is the number of repeating units of galactofuranan, and n is the number of repeating units of glucan. Whereas the polymers from the respective strains differed in the numbers of repeating units of the galactofuranan and glucan moieties and in the number of fatty acid residues, the proposed structure is essentially the same as that reported previously for the macroamphiphile of B. bifidum subsp. pennsylvanicum DSM 20239 by W. Fischer (Eur. J. Biochem. 165:639-646, 1987).
DNA sequences and characterization of four early genes of the tryptophan pathway in Pseudomonas aeruginosa
Authors: Essar D W , Eberly L , Han C Y , Crawford I P
Published: November 2016

Two pairs of related but easily distinguishable genes for the two subunits of anthranilate synthase have been identified in Pseudomonas aeruginosa. These were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance cassette, and returned to the P. aeruginosa chromosome, replacing the wild-type gene. Gene replacement implicated only one of the pairs in tryptophan biosynthesis. This report describes the cloning and sequencing of the tryptophan-related gene pair, designated trpE and trpG, and presents experiments implicating their gene products in tryptophan production. DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also includes trpD and trpC. Complementation of Trp auxotrophs by R-prime plasmids (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983) has shown that a large cluster of pyocin R2 genes is flanked at one end by trpE and the other end by trpDC; the physical map that was obtained shows the distance between trpE and trpDC to be about 25 kilobases. Our restriction map of the trpE and trpGDC regions agrees with data presented by Shinomiya et al.
Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco
Authors: Vanneste J L , Paulin J P , Expert D
Published: November 2016

Erwinia amylovora 1430 was shown to be sensitive to Mu G(-) particles. Infection resulted either in lytic development or in lysogenic derivatives with insertion of the Mu genome at many sites in the bacterial chromosome. We used the Mu d1Bx::Tn9 (lac Apr Cmr) derivative, called Mu dX, to identify mutants affected in pathogenicity and in their ability to induce a hypersensitive reaction (HR) on tobacco plants. Inoculation of 1,400 lysogenic derivatives on apple root calli led to the identification of 12 mutants in three classes: (i) class 1 mutants were nonpathogenic and unable to induce an HR on tobacco plants; (ii) class 2 mutants were nonpathogenic but retained the ability to induce an HR; and (iii) class 3 mutants showed attenuated virulence. Of the 12 mutants, 8 had a single insertion of the Mu dX prophage. For class 1 and 2 mutants, reversion to pathogenicity was concomitant with the loss of the Mu dX prophage. Furthermore, revertants from the class 1 mutants also recovered the ability to induce an HR on tobacco plants. Five of the six class 3 mutants were impaired in exopolysaccharide production. No changes of the envelope structure (lipopolysaccharide and outer membrane proteins) were correlated with differences in pathogenicity. One class 3 mutant did not produce any functional siderophore, suggesting that iron uptake could be involved in pathogenicity.
Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects
Authors: Van Spanning R J , Wansell C , Harms N , Oltmann L F , Stouthamer A H
Published: November 2016

By using synthetic oligonucleotides, the gene encoding soluble cytochrome c550 was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the mature protein was found to be similar to the primary structure of purified cytochrome c550 except for the presence of seven additional amino acid residues at the C terminus. At the N terminus of the primary structure was found an additional stretch of 19 amino acid residues that had the typical features of the signal sequence of the cytochrome. Comparison of the nucleotide sequences of the upstream regions of the P. denitrificans cytochrome c550 gene and bc1 operon revealed three regions with a distinct organization that showed strong similarity. Downstream of the c550 gene was found part of another gene, the deduced amino acid sequence of which showed strong homology with subunit 1 of the cytochrome aa3 oxidase. For gene replacement experiments, the suicide vector pGRPd1 was constructed. The cytochrome c550 gene was inactivated by insertion of a kanamycin resistance gene, and the mutated gene was cloned into this vector. Recombination with the wild-type gene resulted in a mutant strain with an inactivated cytochrome gene. Isolated mutant strains were unable to synthesize the soluble cytochrome, as judged by spectrum analysis and analysis of periplasmic proteins by gel electrophoresis and heme staining. The mutation resulted in a 14% decrease in the growth yield during aerobic heterotrophic growth and in a 40% decrease in the maximum specific growth rate during growth on methylamine. Furthermore, a longer lag phase was observed under both growth conditions. The mutation had no effect on growth yield, maximum specific growth rate, and duration of the lag phase during anaerobic growth in the presence of nitrate. In addition, there was no accumulation of nitrite and nitrous oxide.
Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes
Authors: Cookson B T , Berg D E , Goldman W E
Published: November 2016

The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of interest, providing a useful tool for studying virulence and other important traits of diverse bacterial species.
A cryptic 65-kilobase-pair transposonlike element isolated from Bacteroides uniformis has homology with Bacteroides conjugal tetracycline resistance elements
Authors: Shoemaker N B , Salyers A A
Published: November 2016

A 65-kilobase-pair element, XBU4422, which has some transposonlike characteristics but carries no known antibiotic resistance genes, has been isolated from Bacteroides uniformis 0061. XBU4422 was trapped on Bacteroides-Escherichia coli shuttle vectors during experiments in which one of the conjugal Bacteroides tetracycline resistance (Tcr) elements was being used to mobilize the shuttle vectors to Bacteroides recipients. Results of Southern hybridization experiments showed that XBU4422 is normally integrated in the B. uniformis 0061 chromosome and is found only in some strains. Insertion of XBU4422 in the shuttle vectors was site specific and orientation specific. Nonmobilizable vectors that had acquired XBU4422 became transmissible and could be transferred to Bacteroides or E. coli recipients. In B. uniformis transconjugants, the XBU4422 insertion in the vectors was usually intact, but XBU4422 was always lost in matings with E. coli, Bacteroides thetaiotaomicron, or B. ovatus. The loss of XBU4422 did not visibly alter the vector; in the case of E. coli, the loss of the insertion appeared to be RecA dependent. Although XBU4422 carried no antibiotic resistances, it shared regions of homology with six conjugal Bacteroides Tcr elements; this homology was strongest with the ends of XBU4422. Using a strain of B. thetaiotaomicron that contains no XBU4422-hybridizing sequences, we showed that the ends of XBU4422 were probably reacting with the ends of the Tcr elements. These results provide the first direct evidence that the Tcr elements, like XBU4422, are integrated in the chromosome and that insertion of the least some Tcr elements, such as TcrEmr DOT, is relatively site specific.
Mutants of Streptococcus faecalis sensitive to alkaline pH lack Na(+)-ATPase
Authors: Kakinuma Y , Igarashi K
Published: November 2016

Alkali-sensitive mutants which grow at pH 7.5 but not at pH 9.5 in Na(+)-rich media were isolated from Streptococcus faecalis ATCC 9790. One of the mutants, designated Nak1, lacked activities of both Na(+)-stimulated ATPase and KtrII (active K+ uptake by sodium ATPase). These activities were restored in a spontaneous revertant designated Nak1R. Active sodium extrusion from Nak1 was observed at pH 7.0, which allows the cells to generate a proton potential, but not at pH 9.5, which reverses the proton potential, making it positive. Sodium extrusion at pH 7.0 was inhibited by addition of dicyclohexylcarbodiimide and protonophores. Even at pH 9.5, Nak1 did grow well in Na(+)-poor media. In Na(+)-rich media at pH 7.5, growth of Nak1 but not that of 9790 was severely inhibited by a protonophore. These results indicate that mutant Nak1 lacks sodium ATPase but contains a sodium/proton antiporter and that sodium ATPase is essential for the growth of this organism at high pH in Na(+)-rich conditions.
Immunochemical analysis of lipopolysaccharides from free-living and endosymbiotic forms of Rhizobium leguminosarum
Authors: Sindhu S S , Brewin N J , Kannenberg E L
Published: November 2016

Rhizobium leguminosarum B556 and 8002 differ only with respect to carrying symbiotic plasmids with specificity for Pisum or Phaseolus hosts, respectively. Protease-treated samples derived from free-living cultures of both strains revealed a ladder of lipopolysaccharide (LPS-1) bands after periodate-silver staining of sodium dodecyl sulfate-polyacrylamide gels. These bands were arranged as doublets. After Western (immuno-) blotting, all LPS-1 bands reacted with monoclonal antibody JIM 21, whereas monoclonal antibody MAC 57 reacted only with the upper (slower-migrating) band and monoclonal antibody MAC 114 reacted only with the lower band of each doublet pair. Preparations obtained from bacteroids of Pisum or Phaseolus nodules showed significant differences in the size distribution and antigenicity of LPS. In bacteroids from Phaseolus sp., JIM 21 and MAC 57 each stained a ladder of LPS-1 bands on sodium dodecyl sulfate-polyacrylamide gels which corresponded in mobility to the upper band of each doublet pair seen in free-living cultures. MAC 114 did not react with the LPS from Phaseolus sp.-derived bacteroids. In bacteroids from Pisum sp., only fast-migrating (lower-molecular-weight) forms of LPS-1 could be visualized on gels, but both upper and lower bands of each doublet were still present and could be stained by the appropriate monoclonal antibody, MAC 57 or MAC 114, respectively. Similarly, bacteroids from R. leguminosarum 3841, which nodulates Pisum species, differed with respect to the structure and antigenicity of their LPS-1 from bacteroids of a related strain, B625, which nodulates Phaseolus species. Physiological factors were investigated that could account for these differences between the structures of LPS-1 from free-living cultures of B556 and 8002 and that from bacteroids. The following modifications in growth conditions each tended to reduce the expression of MAC 114 antigen and enhance the expression of MAC 57 antigen: succinate rather than glucose as the carbon source; microaerobic (2.5%, vol/vol) oxygen concentrations; and acidic (pH 5 to 6) culture medium. When all three of these conditions were combined, the LPS-1 that resulted was very similar to that in bacteroids from Pisum nodules. However, it was not possible to reproduce the LPS-1 pattern observed for bacteroids from Phaseolus nodules, which maintained a ladder of LPS bands reacting with MAC 57 antibody.
A chromosomal Agrobacterium tumefaciens gene required for effective plant signal transduction
Authors: Huang M L , Cangelosi G A , Halperin W , Nester E W
Published: November 2016

The vir gene products of Agrobacterium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.
Chromosomal transformation in the cyanobacterium Agmenellum quadruplicatum
Authors: Essich E , Stevens S E , Porter R D
Published: November 2016

Chromosomal transformation of Agmenellum quadruplicatum PR-6 (= Synechococcus sp. strain 7002) was characterized for phenotypic expression, for exposure time to DNA, and for dependence on DNA concentration with regard to Rifr donor DNA. Exponentially growing cells of PR-6 were competent for chromosomal transformation. Competence decreased in cells in the stationary phase of growth or in cells deprived of a nitrogen source. Dark incubation of cells before exposure to donor DNA also decreased competence. Homologous Rifr and Strr DNA and heterologous Escherichia coli W3110 DNA were used in DNA-DNA competition studies, which clearly showed that DNA binding by PR-6 was nonspecific. DNA binding and uptake by PR-6 exhibited single-hit kinetics. Single-stranded DNA failed to transform competent cells of PR-6, and DNA eclipse was not observed, suggesting that double-stranded DNA was the substrate for the binding and uptake reactions during the transformation of PR-6. A significant improvement in transformation frequency was achieved by increasing the nitrate content of the culture medium and by lowering the temperature at which cells were exposed to donor DNA from 39 degrees C (the optimal temperature for growth) to 30 degrees C.
Thioredoxin or glutaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucleotide synthesis
Authors: Russel M , Model P , Holmgren A
Published: November 2016

We have shown previously that Escherichia coli cells constructed to lack both thioredoxin and glutaredoxin are not viable unless they also acquire an additional mutation, which we called X. Here we show that X is a cysA mutation. Our data suggest that the inviability of a trxA grx double mutant is due to the accumulation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in the sulfate assimilation pathway. The presence of excess cystine at a concentration sufficient to repress the sulfate assimilation pathway obviates the need for an X mutation and prevents the lethality of a novel cys+ trxA grx double mutant designated strain A522. Mutations in genes required for PAPS synthesis (cysA or cysC) protect cells from the otherwise lethal effect of elimination of both thioredoxin and glutaredoxin even in the absence of excess cystine. Both thioredoxin and glutaredoxin have been shown to be hydrogen donors for PAPS reductase (cysH) in vitro (M. L.-S. Tsang, J. Bacteriol. 146:1059-1066, 1981), and one or the other of these compounds is presumably essential in vivo for growth on minimal medium containing sulfate as the sulfur source. The cells which lack both thioredoxin and glutaredoxin require cystine or glutathione for growth on minimal medium but maintain an active ribonucleotide reduction system. Thus, E. coli must contain a third hydrogen donor active with ribonucleotide reductase.
Control of Escherichia coli superoxide dismutase (sodA and sodB) genes by the ferric uptake regulation (fur) locus
Authors: Niederhoffer E C , Naranjo C M , Bradley K L , Fee J A
Published: November 2016

The ferric uptake regulation (fur) gene product participates in regulating expression of the manganese- and iron-containing superoxide dismutase genes of Escherichia coli. Examination of beta-galactosidase activity coded from a chromosomal phi(sodA'-'lacZ) fusion suggests that metallated Fur protein acts as a transcriptional repressor of sodA (manganese superoxide dismutase [MnSOD]). Gel retardation assays demonstrate high-affinity binding of pure, Mn2(+)-Fur protein to DNA fragments containing the sodA promoter. These data and the presence of an iron box sequence in its promoter strongly suggest that sodA is part of the iron uptake regulon. An sodB'-'lacZ fusion gene borne on either a low- or high-copy plasmid yielded approximately two- to threefold more beta-galactosidase activity in Fur+ compared with Fur- cells; the levels of activity depended only weakly on the growth phase and did not change during an extended stationary phase. Measurement of FeSOD activity in logarithmic growth phase and in overnight cultures of sodA and fur sodA backgrounds revealed that almost no FeSOD activity was expressed in Fur- strains, whereas wild-type levels were expressed in Fur+ cells. Fur+ and Fur- cells bearing the multicopy plasmid pHS1-4 (sodB+) expressed approximately sevenfold less FeSOD activity in the fur background, and staining of nondenaturing electrophoretic gels indicates that synthesis of FeSOD protein was greatly reduced in Fur- cells. Gel retardation assays show that Mn2(+)-Fur had a significantly higher affinity for the promoter fragment of sodB compared with that of random DNA sequences but significantly lower than for the promoter fragment of sodA. These observations suggest that the apparent positive regulation of sodB does not result exclusively from a direct interaction of holo (metallated) Fur itself with the sodB promoter. Nevertheless, the sodB gene also appears to be part of the iron uptake regulon but not in the classical manner of Fe-dependent repression.
Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity
Authors: Bustos S A , Schaefer M R , Golden S S
Published: November 2016

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes which encode two forms of the D1 protein of photosystem II. Experiments using psbA-lacZ translational fusions and Western blot (immunoblot) analysis have shown that the psbA genes respond differently to changes in light intensity, altering the ratio of the two forms of D1 in the thylakoid membrane. Each gene produces a 1.2-kilobase (kb) mRNA. A probe specific for psbAII transcripts also identified a 1.6-kb mRNA which starts 419 base pairs upstream of the 5' end of the 1.2-kb species and overlaps the entire 1.2-kb transcript. This 419-base-pair region includes an open reading frame (ORF1) of 114 amino acids. We investigated the effects of changes in light intensity on psbAII transcript levels in a series of light shift experiments in the wild-type Synechococcus sp. and in AMC084, a mutant which does not produce the 1.6-kb transcript. After exposure to high light intensities for 15 min, the level of the 1.2-kb psbAII transcript increased in both strains. This transcript was not detected in either strain after transfer to low light intensity. The psbAIII transcript showed the same pattern of response as the 1.2-kb psbAII transcript, whereas the 1.6-kb psbAII transcript was unaffected by different light intensities. The psbAI transcript levels responded oppositely to those of psbAII and psbAIII. These data, considered along with previous results obtained by using lacZ translational gene fusions, indicate that the response of psbA genes to changes in light intensity is controlled primarily at the transcriptional level.
Roles for enteric d-type cytochrome oxidase in N2 fixation and microaerobiosis
Authors: Hill S , Viollet S , Smith A T , Anthony C
Published: November 2016

Escherichia coli strains that lacked the d-type cytochrome oxidase, the terminal oxidase with a high affinity for O2, grew anaerobically as well as the wild type did and were not impaired in the ability to evolve H2 from either glucose or formate. The anaerobic synthesis and activity of nitrogenase in transconjugants of these strains carrying Klebsiella pneumoniae nif genes were also normal. However, the behavior towards O2 of anaerobically grown bacteria lacking the d-type oxidase differed from that of the wild type in the following ways: the potential O2 uptake was lower, H2 evolution and nitrogenase activity supported by fermentation were more strongly inhibited by O2, and microaerobic O2-dependent nitrogenase activity in the absence of a fermentable carbon source did not occur. These results show that the d-type oxidase serves two functions in enteric bacteria--to conserve energy under microaerobic conditions and to protect anaerobic processes from inhibition by O2.
Identification of a new gene, molR, essential for utilization of molybdate by Escherichia coli
Authors: Lee J H , Wendt J C , Shanmugam K T
Published: November 2016

A mutation in a new gene, molR, prevented the synthesis in Escherichia coli of molybdoenzymes, including the two formate dehydrogenase isoenzymes, nitrate reductase and trimethylamine-N-oxide reductase. This phenotype was suppressed by supplementing the media with molybdate. Thus, the molR mutant was phenotypically similar to previously described chlD mutants, thought to be defective in molybdate transport. The molR gene is located at 65.3 min in the E. coli chromosome, in contrast to the chlD gene, which maps at 17 min and thus can be readily distinguished. The molR gene is also cotransducible with a hitherto unidentified gene essential for the production of 2-oxoglutarate from isocitrate, designated icdB (located at 66 min). The molR mutant strain SE1100 also failed to produce the hydrogenase component of formate hydrogenlyase (HYD3) in molybdate-unsupplemented media. The amount of molybdate required by strain SE1100 for the production of parental levels of formate hydrogenlyase activity was dependent on the growth medium. In Luria-Bertani medium, this value was about 100 microM, and in glucose-minimal medium, 1.0 microM was sufficient. In low-sulfur medium, this value decreased to about 50 nM. The addition of sulfate or selenite increased the amount of molybdate needed for the production of formate hydrogenlyase activity. These data suggest that in the absence of the high-affinity molybdate transport system, E. coli utilizes sulfate and selenite transport systems for transporting molybdate, preferring sulfate transport over the selenite transport system.
Cloning of genes responsible for acetic acid resistance in Acetobacter aceti
Authors: Fukaya M , Takemura H , Okumura H , Kawamura Y , Horinouchi S , Beppu T
Published: November 2016

Five acetic acid-sensitive mutants of Acetobacter aceti subsp. aceti no. 1023 were isolated by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Three recombinant plasmids that complemented the mutations were isolated from a gene bank of the chromosome DNA of the parental strain constructed in Escherichia coli by using cosmid vector pMVC1. One of these plasmids (pAR1611), carrying about a 30-kilobase-pair (kb) fragment that conferred acetic acid resistance to all five mutants, was further analyzed. Subcloning experiments indicated that a 8.3-kb fragment was sufficient to complement all five mutations. To identify the mutation loci and genes involved in acetic acid resistance, insertional inactivation was performed by insertion of the kanamycin resistance gene derived from E. coli plasmid pACYC177 into the cloned 8.3-kb fragment and successive integration into the chromosome of the parental strain. The results suggested that three genes, designated aarA, aarB, and aarC, were responsible for expression of acetic acid resistance. Gene products of these genes were detected by means of overproduction in E. coli by use of the lac promoter. The amino acid sequence of the aarA gene product deduced from the nucleotide sequence was significantly similar to those of the citrate synthases (CSs) of E. coli and other bacteria. The A. aceti mutants defective in the aarA gene were found to lack CS activity, which was restored by introduction of a plasmid containing the aarA gene. A mutation in the CS gene of E. coli was also complemented by the aarA gene. These results indicate that aarA is the CS gene.
A mutation that enhances synthesis of sigma 32 and suppresses temperature-sensitive growth of the rpoH15 mutant of Escherichia coli
Authors: Yano R , Nagai H , Shiba K , Yura T
Published: November 2016

The rpoH15 mutant cannot grow at or above 34 degrees C, because it produces an altered sigma 32 protein that is largely deficient in the transcription of the heat shock genes. Extragenic suppressor mutations (suhB) located at 55 min on the Escherichia coli chromosome endowed the mutant cell with the ability to grow at 40 degrees C and the inability to grow at 25 degrees C. One such mutation (suhB2), studied in detail, markedly enhanced the rate of sigma 32 synthesis and the rpoH mRNA level during steady-state growth at 37 to 40 degrees C but little affected the cellular content of sigma 32 or the induction of heat shock proteins. In the isogenic rpoH+ strain, neither sigma 32 synthesis nor the rpoH mRNA level was enhanced by the suhB suppressor. Furthermore, expression of the rpoH-lacZ gene fusion, but not the operon fusion, was much higher in the suhB mutant than in the wild type or the suhB rpoH+ strain, indicating that suhB affects rpoH expression primarily at the level of translation. suhB probably acts to increase sigma 32 synthesis by affecting the regulatory circuit of rpoH expression or by modulating certain parameters in protein synthesis. Consistent with these findings, overproduction of the mutant (rpoH15) sigma 32 by multicopy plasmid enabled the rpoH15 or delta rpoH (deletion) mutant to grow at up to 40 degrees C. Plasmids containing an E. coli DNA segment of 1.0 kilobase could complement the cold-sensitive phenotype of the suhB2 mutant. Nucleotide sequence analysis revealed that the segment contained an open reading frame encoding a protein of 29,128 daltons.
A sulfur- and tyramine-regulated Klebsiella aerogenes operon containing the arylsulfatase (atsA) gene and the atsB gene
Authors: Murooka Y , Ishibashi K , Yasumoto M , Sasaki M , Sugino H , Azakami H , Yamashita M
Published: November 2016

The structural gene for arylsulfatase (atsA) of Klebsiella aerogenes was cloned into a pKI212 vector in Escherichia coli. Deletion analysis showed that the atsA gene with the promoter region was located within a 3.2-kilobase cloned segment. In E. coli cells which carried the plasmid, the synthesis of arylsulfatase was repressed by various sources of sulfur; the repression was relieved, in each case, by tyramine. Transfer of the plasmid into atsA or constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsA but not of atsR. The nucleotide sequence of the 3.2-kilobase fragment was determined. Two open reading frames, the atsA gene and an unknown gene (atsB), were found. These are located between a potential promoter and a transcriptional terminator sequence. Deletion analysis suggests that atsB is a potential positive factor for the regulation of arylsulfatase. Analysis of the amino acid sequences of the first 13 amino acids from the N terminus of the purified secreted arysulfatase agrees with that of the nucleotide sequence of atsA. The leader peptide extends over 20 amino acids and has the characteristics of a signal sequence. Primer extension mapping of transcripts generated in vivo suggests that the synthesis of mRNA starts at a site 31 or 32 bases upstream from the ATG initiation codon of the atsB gene. By Northern (RNA) blot analysis of the transcripts induced by tyramine, we found a 2.7-kilobase transcript which is identical in size to the total sequence of the atsB and atsA genes. Thus, the ats operon is composed of two cistrons and is regulated by sulfur and tyramine.