bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a CO-dependent O-demethylating enzyme system from the acetogen Clostridium thermoaceticum
Authors: Wu Z R , Daniel S L , Drake H L
Published: November 2016

An inducible O-demethylating enzyme system was characterized from Clostridium thermoaceticum cultivated at the expense of syringate. Glucose and methanol, but not CO, partially repressed its expression. Induced whole cells catalyzed the carbon monoxide (CO)-dependent O demethylation of methoxylated aromatic compounds with the concomitant formation of acetate. Pyruvate and, to a lesser extent, H2-CO2 could replace CO in these reactions. KCN inhibited pyruvate-dependent activity but not the CO-dependent activity. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide, the protonophore carbonyl cyanide m-chlorophenylhydrazone, and methyl viologen did not appreciably inhibit O demethylation by induced cells, whereas Triton X-100 was inhibitory. The enzyme system appeared to convert syringate sequentially to 5-hydroxyvanillate and gallate. The proposed overall reaction stoichiometry was as follows: syringate + 2CO + 2H2O----gallate + 2 acetates. Growth-supportive methoxylated aromatic compounds were O demethylated by syringate-cultivated cells and inhibitory to syringate O demethylation.
Anaerobic metabolism of phthalate and other aromatic compounds by a denitrifying bacterium
Authors: Nozawa T , Maruyama Y
Published: November 2016

The anaerobic metabolism of phthalate and other aromatic compounds by the denitrifying bacterium Pseudomonas sp. strain P136 was studied. Benzoate, cyclohex-1-ene-carboxylate, 2-hydroxycyclohexanecarboxylate, and pimelate were detected as predominant metabolic intermediates during the metabolism of three isomers of phthalate, m-hydroxybenzoate, p-hydroxybenzoate, and cyclohex-3-ene-carboxylate. Inducible acyl-coenzyme A synthetase activities for phthalates, benzoate, cyclohex-1-ene-carboxylate, and cyclohex-3-ene-carboxylate were detected in the cells grown on aromatic compounds. Simultaneous adaptation to these aromatic compounds also occurred. A similar phenomenon was observed in the aerobic metabolism of aromatic compounds by this strain. A new pathway for the anaerobic metabolism of phthalate and a series of other aromatic compounds by this strain was proposed. Some properties of the regulation of this pathway were also discussed.
Complementation and hybridization evidence for additional families of type I DNA restriction and modification genes in Salmonella serotypes
Authors: Ryu J , Rajadas P T , Bullas L R
Published: November 2016

Of eight Salmonella, serB-linked hsd genes for the restriction and modification of DNA transferred to Escherichia coli/Salmonella hybrids, only two--those with SM and ST (S. muenchen and S. thompson, respectively) specificities--may have weakly complemented rSB- and none complemented rK-. An A-specific DNA probe failed to hybridize to HindIII-restricted fragments of each of the hybrids, but an SB (S. typhimurium)-specific probe hybridized to DNA from the hybrid with ST specificity. These results indicate that additional families of the type I hsd genes may exist.
High-frequency transformation of Rhizobium meliloti
Authors: Courtois J , Courtois B , Guillaume J
Published: November 2016

Transformation of R factor RP4 and its derivative pRK290 from Escherichia coli to Rhizobium meliloti is reported. The efficiency of transformation was in the range of 10(-5) per viable cell. In addition, chromosomal DNA prepared from one R. meliloti strain resistant to streptomycin was transferred to the isoleucine-valine-requiring mutant susceptible to streptomycin.
Control of bacterial alkaline phosphatase synthesis and variation in an Escherichia coli K-12 phoR mutant by adenyl cyclase, the cyclic AMP receptor protein, and the phoM operon
Authors: Wanner B L , Wilmes M R , Young D C
Published: November 2016

Mutant phoR cells show a clonal variation phenotype with respect to bacterial alkaline phosphatase (BAP) synthesis. BAP clonal variation is characterized by an alternation between a Bap+ and Bap- phenotype. The switching is regulated by the phoM operon and the presence of glucose; the pho-510 mutant form of the phoM operon abolishes both BAP clonal variation and the effect of glucose (B.L. Wanner, J. Bacteriol. 169:900-903, 1987). In this paper we show that a mutation of the adenyl cyclase (cya) and the cyclic AMP receptor protein (crp) gene also abolish BAP clonal variation; either simultaneously reduces the amount of BAP made in phoR mutants. Also, the pho-510 mutation is epistatic; it increases BAP synthesis in delta cya phoR and delta crp phoR mutants. These data are consistent with the wild-type phoM operon having a negative, as well as a positive, regulatory role in gene expression. Furthermore, the data suggest that adenyl cyclase and Crp indirectly regulate BAP synthesis in a phoR mutant via an interaction with the phoM operon or its gene products. However, phoM operon expression was unaffected when tested with phoM operon lacZ transcriptional fusions. In addition, the switching Bap phenotype was not associated with an alternation in phoM operon expression.
Mutation in the phosphoribosylpyrophosphate synthetase gene (prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli
Authors: Hove-Jensen B
Published: November 2016

A mutant of Escherichia coli harboring a temperature-labile phosphoribosylpyrophosphate (PRPP) synthetase was characterized. Despite the lack of a detectable PRPP pool or PRPP synthetase activity at 40 degrees C, the strain was fully viable at this temperature as long as guanosine, uridine, histidine, tryptophan, and nicotinamide mononucleotide were all added to the growth medium. Viability of the strain was dependent upon mutations in genes of the nucleoside salvage pathways that improved the utilization of exogenous nucleosides. The properties of the strain are those expected of a PRPP-less strain and suggest that PRPP synthetase is dispensable for E. coli.
New loci required for Streptomyces coelicolor morphological and physiological differentiation
Authors: Champness W C
Published: November 2016

Streptomyces coelicolor colonies differentiate both morphologically, producing aerial spore chains, and physiologically, producing antibiotics as secondary metabolites. Single mutations, which block both aspects of differentiation, define bld (bald colony) genes. To identify new bld genes, mutagenized colonies were screened for blocks in the earliest stage of sporulation, the formation of aerial mycelia, and blocks in antibiotic synthesis. The mutations in 12 mutants were mapped; in each strain, the pleiotropic phenotype was due to a single mutation. Seven of the strains contained mutations in known bld loci, bldA and bldB. Three strains contained mutations in a new locus, bldG, and two contained mutations in another new locus, bldH. Like the previously defined bldA mutants, the bldG and bldH mutants were developmentally blocked on glucose. On a variety of carbon sources whose utilization was subject to glucose repression, the developmental blocks were partially relieved for bldG (and bldA) mutants and fully relieved for bldH mutants. These results are compatible with an hypothesis which suggests that there are two alternative controls on S. coelicolor differentiation, one of which is glucose repressible.
Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli
Authors: Speer B S , Salyers A A
Published: November 2016

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.
Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens
Authors: Veluthambi K , Ream W , Gelvin S B
Published: November 2016

Agrobacterium tumefaciens transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of A. tumefaciens A348 with regenerating tobacco leaf protoplasts, six distinct single-stranded T-DNA molecules (T strands) were generated in addition to double-stranded T-DNA border cleavages which we have previously reported (K. Veluthambi, R.K. Jayaswal, and S.B. Gelvin, Proc. Natl. Acad. Sci. USA 84:1881-1885, 1987). The T region of an octopine-type Ti plasmid has four border repeats delimiting three T-DNA regions defined as T left (TL), T center (TC), and T right (TR). The six T strands generated upon induction corresponded to the TL, TC, TR, TL + TC, TC + TR, and TL + TC + TR regions, suggesting that the initiation and termination of T-strand synthesis can occur at each of the four borders. Most TL + TC + TR T-strand molecules corresponded to the top T-DNA strand, whereas the other five T strands corresponded to the bottom T-DNA strand. Generation of T strands required the virA, virG, and virD operons. Extra copies of vir genes, harbored on cosmids within derivatives of A. tumefaciens A348, enhanced production of T strands. The presence of right and left border repeats in their native orientation is important for the generation of full-length T strands. When a right border repeat was placed in the opposite orientation, single-stranded T-DNA molecules that corresponded to the top strand were generated. Deletion of overdrive, a sequence that flanks right border repeats and functions as a T-DNA transmission enhancer, reduced the level of T-strand generation. Induction of A. tumefaciens cells by regenerating tobacco protoplasts increased the copy number of the Ti plasmid relative to the bacterial chromosome.
Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu
Authors: Bourret R B , Fox M S
Published: November 2016

The possible outcomes of infection of Escherichia coli by bacteriophage Mu include lytic growth, lysogen formation, nonlysogenic surviving cells, and perhaps simple killing of the host. The influence of various parameters, including host himA and himD mutations, on lysogeny and cell survival is described. Mu does not grow lytically in or kill him bacteria but can lysogenize such hosts. Mu c+ lysogenizes about 8% of him+ bacteria infected at low multiplicity at 37 degrees C. The frequency of lysogens per infected him+ cell diminishes with increasing multiplicity of infection or with increasing temperature over the range from 30 to 42 degrees C. In him bacteria, the Mu lysogenization frequency increases from about 7% at low multiplicity of infection to approach a maximum where most but not all cells are lysogens at high multiplicity of infection. Lysogenization of him hosts by an assay phage marked with antibiotic resistance is enhanced by infection with unmarked auxiliary phage. This helping effect is possible for at least 1 h, suggesting that Mu infection results in formation of a stable intermediate. Mu immunity is not required for lysogenization of him hosts. We argue that in him bacteria, all Mu genomes which integrate into the host chromosome form lysogens.
Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate
Authors: Geissler J F , Harwood C S , Gibson J
Published: November 2016

A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 mumol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg2+ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 microM for benzoate, 2 to 3 microM for ATP, and 90 to 120 microM for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.
Effects of oxygen stress on membrane functions in Escherichia coli: role of HPI catalase
Authors: Farr S B , Touati D , Kogoma T
Published: November 2016

Different conditions of oxidative stress were used to study their effects on membrane transport in Escherichia coli K-12. The oxidizing conditions included H2O2, plumbagin (a redox cycling compound that generates superoxide radicals [O2-]), and increased partial pressure of oxygen. Both superoxide radical-generating conditions and H2O2 treatments were found to cause a rapid decrease in proton motive force-dependent and -independent transport. H2O2-pretreated cells had the ability to rapidly recover both proton motive force-dependent and -independent transport. The induction required transcription and translation and was dependent on oxyR+ and katG+, providing evidence that these genes play crucial roles in the rapid recovery of transport. The effects of oxidatively induced loss of proton motive force on cell growth and macromolecular synthesis were also investigated.
The Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor
Authors: Peterson K R , Ossanna N , Mount D W
Published: November 2016

LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background. LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor. lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile).
Proteus mirabilis urease: genetic organization, regulation, and expression of structural genes
Authors: Jones B D , Mobley H L
Published: November 2016

Proteus mirabilis, a cause of serious urinary tract infection, produces urease, an important virulence factor for this species. The enzyme hydrolyzes urea to CO2 and NH3, which initiates struvite or apatite stone formation. Genes encoding urease were localized on a P. mirabilis chromosomal DNA gene bank clone in Escherichia coli by deletion analysis, subcloning, Bal31 nuclease digestion, transposon Tn5 mutagenesis, and in vitro transcription-translation. A region of DNA between 4.0 and 5.4 kilobases (kb) in length was necessary for urease activity and was located within an 18.5-kb EcoRI fragment. The operon was induced by urea and encoded a multimeric, cytoplasmic enzyme comprising subunit polypeptides of 8,000, 10,000, and 73,000 daltons that were encoded by a single polycistronic mRNA and transcribed in that order. Seventeen urease-negative transposon insertions were isolated that synthesized either none of the structural subunit polypeptides, the 8,000-dalton polypeptide alone, or both the 8,000- and 10,000-dalton subunit polypeptides. The molecular weight of the native enzyme was estimated to be 212,000 by Superose-6 chromatography. Homologous sequences encoding the urease of Providencia stuartii synthesized subunit polypeptides of similar sizes and showed a similar genetic arrangement. However, restriction maps of the operons from the two species were distinct, indicating significant divergence.
Characterization of the upstream region of the formate dehydrogenase operon of Methanobacterium formicicum
Authors: Patel P S , Ferry J G
Published: November 2016

The fdhA and fdhB genes of Methanobacterium formicicum, which code for the alpha and beta subunits of formate dehydrogenase, were cotranscribed as part of a large transcript. By using Northern (RNA) gel blot analysis, the transcription start site was located within a 1.6-kilobase BglII-NcoI fragment 4.3 kilobases upstream from the fdhA gene. The precise transcription start site within the fragment was determined with the aid of primer extension analysis and S1 nuclease protection studies. A putative promoter sequence for structural genes of methanogenic archaebacteria is proposed based on a comparison of DNA sequences of the upstream region of methanogen operons for which transcription initiation sites are known. Comparison of the DNA sequence of the upstream region of the fdh operon of M. formicicum with the sequence upstream of the fdhF gene of Escherichia coli revealed regions of considerable identity.
Evolutionary relationships among cyanobacteria and green chloroplasts
Authors: Giovannoni S J , Turner S , Olsen G J , Barns S , Lane D J , Pace N R
Published: November 2016

The 16S rRNAs from 29 cyanobacteria and the cyanelle of the phytoflagellate Cyanophora paradoxa were partially sequenced by a dideoxynucleotide-terminated, primer extension method. A least-squares distance matrix analysis was used to infer phylogenetic trees that include green chloroplasts (those of euglenoids, green algae, and higher plants). The results indicate that many diverse forms of cyanobacteria diverged within a short span of evolutionary distance. Evolutionary depth within the surveyed cyanobacteria is substantially less than that separating the major eubacterial taxa, as though cyanobacterial diversification occurred significantly after the appearance of the major eubacterial groups. Three of the five taxonomic sections defined by Rippka et al. (R. Rippka, J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier, J. Gen. Microbiol. 111:1-61, 1979) (sections II [pleurocapsalean], IV [heterocystous, filamentous, nonbranching], and V [heterocystous, filamentous, branching]) are phylogenetically coherent. However, the other two sections (I [unicellular] and III [nonheterocystous, filamentous]) are intermixed and hence are not natural groupings. Our results not only support the conclusion of previous workers that the cyanobacteria and green chloroplasts form a coherent phylogenetic group but also suggest that the chloroplast lineage, which includes the cyanelle of C. paradoxa, is not just a sister group to the free-living forms but rather is contained within the cyanobacterial radiation.
The product of the F plasmid transfer operon gene, traF, is a periplasmic protein
Authors: Wu J H , Kathir P , Ippen-Ihler K
Published: November 2016

The products of clones carrying the F plasmid transfer operon gene, traF, were analyzed. Proteins expressed in maxicells were labeled with [35S]methionine and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Clones carrying the wild-type traF gene expressed two polypeptide products that were not products of clones containing the traF13 amber mutation. These migrated with apparent molecular weights (Ma) of 27,000 and 25,000. A pulse-chase experiment suggested that the larger product was a precursor of the smaller one. In the presence of ethanol, the Ma-27,000 polypeptide accumulated and the Ma-25,000 product was not expressed. These results indicated that the traF protein undergoes proteolytic processing associated with export. Cell fractionation experiments further indicated that the greatest concentration of the mature (Ma 25,000) TraF protein was located in the periplasm. The DNA sequence of traF and the position of the transition mutation in traF13 DNA were also determined. Sequence analysis suggested that traF would be expressed as a 247-amino-acid, Mr-28,006 polypeptide. The 19 amino acids at the amino terminus of this polypeptide appear to constitute a typical membrane leader peptide, while the remainder of the molecule (Mr 25,942) is predicted to be primarily hydrophilic in character.
A novel outer-membrane-associated protease in Escherichia coli
Authors: Sugimura K , Higashi N
Published: November 2016

Human gamma interferon produced by recombinant Escherichia coli was degraded by endogenous protease after cell disruption. Specific cleavages took place at the center of two pairs of basic amino acids (Lys-131-Arg-132 and Arg-142-Arg-143) in the C-terminal region, giving rise to products with molecular weights of 17,500 and 16,000. The proteolytic activity was associated with the outer membrane of E. coli. It was insensitive to the protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, tosyl-L-lysine chloro-methyl ketone, EDTA, and p-chloromercuribenzoate. Benzamidine and the bivalent cations Zn2+ and Cu2+ inhibited the activity. Dynorphin A(1-13) (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys) was a good substrate and was preferentially cleaved at the center of Arg-6-Arg-7. Neither the amino nor carboxyl sides of Arg-9 and Lys-11 were digested. These results indicate that the protease specifically cleaves the peptide bond between consecutive basic residues and therefore is different from the known membrane enzymes, proteases IV, V, and VI. We have designated this new enzyme protease VII.
Physiological regulation of Paracoccus denitrificans methanol dehydrogenase synthesis and activity
Authors: de Vries G E , Harms N , Maurer K , Papendrecht A , Stouthamer A H
Published: November 2016

An enzyme-linked immunosorbent assay and a whole-cell activity assay were developed which allowed detection of methanol dehydrogenase (MDH) of Paracoccus denitrificans with increased sensitivity. By these methods, it was shown that MDH was not induced by its natural substrate, methanol. Relief from a catabolite repression-like mechanism seemed responsible for low-level MDH synthesis, while product induction was the hypothesized mechanism for synthesis of high amounts of MDH. In the latter process, formaldehyde may play an important role as effector. For a variety of culture conditions, inconsistencies were observed in the relation between amounts of MDH protein synthesized and enzyme activities measured in vitro. Regulation of pyrrolo-quinoline-quinone biosynthesis or a modulation of its incorporation and stability in MDH may constitute an overriding mechanism to ensure a correct tuning between metabolic rates of methanol consumption and the required methanol oxidation rates.
Cloning and nucleotide sequence of a gene for Actinomyces naeslundii WVU45 type 2 fimbriae
Authors: Yeung M K , Cisar J O
Published: November 2016

A genomic library of Actinomyces naeslundii WVU45 DNA in Escherichia coli was screened for antigen expression with rabbit antibody against A. naeslundii fimbriae. Western blotting (immunoblotting) of one recombinant clone carrying a 13.8-kilobase-pair insert revealed a 59-kilodalton (kDa) immunoreactive protein. A protein of similar electrophoretic mobility was detected from the isolated fimbrial antigen. Expression of the 59-kDa cloned protein in E. coli was directed by a promoter from the insert. The DNA sequence of the subunit gene was determined, and an open reading frame of 1,605 nucleotides was identified which was preceded by a putative ribosome-binding site and followed by two inverted repeats of 14 and 17 nucleotides, respectively. The reading frame encoded a protein of 534 amino acids (calculated molecular weight, 57,074), and the N-terminal sequence resembled that of a signal peptide. The presence of a 32-amino-acid signal peptide was indicated by amino-terminal sequencing of the fimbriae from A. naeslundii. The sequence, as determined by Edman degradation, was identical to that deduced from the DNA sequence beginning at predicted residue 33 of the latter sequence. Moreover, the amino acid composition of the predicted mature protein was similar to that of the isolated fimbriae from A. naeslundii. Thus, the cloned gene encodes a subunit of A. naeslundii fimbriae.
Genetic evidence for modulation of the activator by two regulatory proteins involved in the exogenous induction of phosphoglycerate transport in Salmonella typhimurium
Authors: Jiang S Q , Yu G Q , Li Z G , Hong J S
Published: November 2016

Previous work from this laboratory has identified in a fragment of DNA, cloned from Salmonella typhimurium, two genes involved in the exogenous induction of phosphoglycerate transport. These two genes, the transporter gene, pgtP, and the activator gene, pgtA, are closely linked physically; they are only 3.4 kilobases apart. In the accompanying paper, we describe the determination of the nucleotide sequence of this 3.4-kilobase DNA segment and show that this segment contains two genes, pgtB and pgtC, encoding two polypeptides of 593 and 397 amino acid residues, respectively. This paper presents an analysis of the effects of insertions and deletions in pgtBC on the expression of pgtP gene and on the expression of lacZ fused to the pgtP gene. The results indicate that both pgtBC genes are necessary for expression of the pgtP gene. Strikingly, deletion of both genes resulted in a constitutive phenotype, suggesting that PgtB and PgtC polypeptides modulate PgtA activity. The expression of the pgtP gene appears to be regulated by the pgtA gene product, which acts as an activator. A model of induction is proposed in which the central feature is the interaction of the three regulatory proteins in the membrane such that the activity of the activator (PgtA) is subject to modulation by the binding of an inducer.
Chlamydial gene encoding a 70-kilodalton antigen in Escherichia coli: analysis of expression signals and identification of the gene product
Authors: Sardinia L M , Engel J N , Ganem D
Published: November 2016

In an attempt to identify chlamydial genes whose native promoters allow them to be expressed in Escherichia coli, we isolated and characterized a chlamydial gene identified by screening a library of chlamydial DNA with antichlamydial antibodies. This gene encodes a 70-kilodalton immunoreactive polypeptide in E. coli hosts. Sequence analysis of the 5' portion of the gene identified its product as the chlamydial homolog of the E. coli ribosomal protein S1. The site of transcription initiation of the mRNA in chlamydiae was determined, and its putative promoter regions were identified. These regions apparently do not function efficiently in E. coli; in vitro transcripts generated by using E. coli RNA polymerase did not start at the authentic chlamydial initiation site. Several in vitro transcripts both larger and smaller than the authentic transcript were seen; presumably, these transcripts result from adventitious promoterlike elements in adjacent chlamydial DNA and may be responsible for the expression of the gene in E. coli.
Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase
Authors: Mat-Jan F , Alam K Y , Clark D P
Published: November 2016

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.
Cloning and identification of bacteriophage T4 gene 2 product gp2 and action of gp2 on infecting DNA in vivo
Authors: Lipinska B , Rao A S , Bolten B M , Balakrishnan R , Goldberg E B
Published: November 2016

We sequenced bacteriophage T4 genes 2 and 3 and the putative C-terminal portion of gene 50. They were found to have appropriate open reading frames directed counterclockwise on the T4 map. Mutations in genes 2 and 64 were shown to be in the same open reading frame, which we now call gene 2. This gene codes for a protein of 27,068 daltons. The open reading frame corresponding to gene 3 codes for a protein of 20,634 daltons. Appropriate bands on polyacrylamide gels were identified at 30 and 20 kilodaltons, respectively. We found that the product of the cloned gene 2 can protect T4 DNA double-stranded ends from exonuclease V action.
A 20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli
Authors: Adams L F , Visick J E , Whiteley H R
Published: November 2016

The 27-kilodalton (kDa) mosquitocidal protein gene from Bacillus thuringiensis subsp. israelensis has been cloned as a 10-kilobase (kb) HindIII fragment from plasmid DNA; efficient expression in Escherichia coli KM1 depends on a region of DNA located approximately 4 kb upstream (K. McLean and H. R. Whiteley, J. Bacteriol. 169:1017-1023, 1987). We have cloned the upstream DNA region and show that it contains a complete open reading frame (ORF) encoding a protein with a molecular mass of 19,584 Da. Sequencing of adjacent stretches of DNA revealed two partial ORFs: one has 55.2% identity in an overlap of 319 amino acids to the putative transposase of IS231 of B. thuringiensis subsp. thuringiensis, and the other, a 78-codon partial ORF, may be the carboxyl terminus of the 67-kDa protein previously observed in maxicells of strain KM1. A 0.8-kb fragment containing only the 20-kDa protein gene greatly enhanced the expression of the 27-kDa protein in E. coli. The introduction of nonsense codons into the 20-kDa protein gene ORF abolished this effect, indicating that the gene product, not the mRNA or DNA, is required for the enhancement. The effect of the 20-kDa protein gene on various fusions of lacZ to the 27-kDa protein gene suggests that the 20-kDa protein acts after the initiation of translation of the 27-kDa protein gene.
Cloning and characterization of a cluster of linked Bacillus subtilis late competence mutations
Authors: Albano M , Dubnau D A
Published: November 2016

We characterized a segment of chromosomal DNA from Bacillus subtilis that was required for the development of genetic competence. The chromosomal DNA was cloned from a group of genetically linked and phenotypically similar Tn917lac insertion mutants deficient in competence. This cluster of mutations defined the comG locus. Chromosomal DNA flanking each of the six insertions was cloned. Restriction maps of the cloned plasmids revealed that their chromosomal inserts consisted of overlapping fragments. These data, together with Southern blots of chromosomal DNA from the comG mutants, showed that the six Tn917lac comG insertions occurred in the following order: comG12, comG39, comG412, comG107, comG56, and comG210. Expression of the comG Tn917lac insertions was from a promoter located upstream from the first insertion, comG12. This was determined genetically and by low-resolution S1 nuclease mapping of the 3' terminus. The comG region spanned about 5 kilobase pairs, based on low-resolution S1 nuclease mapping of the transcription terminator and Northern blotting. The comG12 mutation had a partial epistatic effect on the expression of one other com locus, comE, but none of the other comG mutations affected expression of this or any other com gene tested. Based on these conclusions, and on its size and phenotype, the comG locus must be organized as a polycistronic operon that is subject to competence-specific regulation.
New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division
Authors: Ishino F , Jung H K , Ikeda M , Doi M , Wachi M , Matsuhashi M
Published: November 2016

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.
Molecular analysis of lcrGVH, the V antigen operon of Yersinia pestis
Authors: Price S B , Leung K Y , Barve S S , Straley S C
Published: November 2016

The lcrGVH operon of plasmid pCD1 in Yersinia pestis KIM encodes the virulence-associated V antigen, the regulatory protein LcrH, and LcrG, a protein of undefined function. In this study we sequenced lcrGVH and analyzed it for transcription initiation sites. There were three open reading frames within the sequence, 288, 981, and 507 bases in length, which could encode proteins with molecular weights and isoelectric points corresponding to those of LcrG, LcrV (V antigen), and LcrH, respectively. The predicted LcrV protein lacked an N-terminal signal sequence; however, an internal signallike sequence was present. An Escherichia coli-like promoter consensus sequence was detected upstream from lcrG. Primer extension analysis showed that (i) the transcriptional start site for lcrGVH was spaced only three bases upstream from the nearest ATG potential start site, raising the possibility that Y. pestis may use an alternate initiation codon for the V operon; (ii) there was much more primer-extended product in yersiniae grown in the absence of Ca2+ than in its presence, showing for the first time that lcrGVH is regulated at the transcriptional level by Ca2+; (iii) no separate lcrV initiation was detected, indicating that the V antigen is expressed from messages initiating at lcrG; and (iv) a non-Ca2+-regulated transcriptional start site was found upstream from lcrH, suggesting that the LcrH protein is expressed constitutively. However, two-dimensional gel analysis showed that net LcrH expression was regulated by Ca2+. We propose that lcrH lies within two differentially regulated operons, its own and lcrGVH.
Identification and sequence of the gene for abequose synthase, which confers antigenic specificity on group B salmonellae: homology with galactose epimerase
Authors: Wyk P , Reeves P
Published: November 2016

The O antigen of Salmonella group B strains contains the sugar abequose, whereas those from group A and D strains contain paratose or tyvelose in its place. This is the essential difference between these Salmonella groups. Only the final step in the biosynthesis of abequose differs from that of paratose, and the abequose confers on group B strains their specific O4 antigen. The gene, rfbJ, encoding the enzyme abequose synthase for this last specific step has been cloned, identified, and sequenced and has been shown to function in group A and D strains to make them O4+. This one gene thus differentiates group B from group A or group D salmonellae. The enzyme abequose synthase appears to be related to galactose epimerase, and the significance of this is discussed. The rfbJ gene and adjacent DNA is of much lower G+C content than is usual for salmonellae, indicating that the region did not originate in a salmonella but was transferred from outside.
Identification and sequence of rfbS and rfbE, which determine antigenic specificity of group A and group D salmonellae
Authors: Verma N , Reeves P
Published: November 2016

Salmonella group A, group B, and group D strains have paratose, abequose, and tyvelose, respectively, as the immunodominant sugar in their O antigens, which are otherwise identical; only the final steps differ in the biosynthetic pathways of these sugars. The gene rfbJ from a group B strain, encoding abequose synthase, the final and only unique step in the biosynthesis of CDP-abequose, has been cloned and sequenced (P. Wyk and P. Reeves, J. Bacteriol. 171:5687-5693, 1989). In this study, we locate and sequence rfbS and rfbE from serovars typhi and paratyphi, representative of groups A and D. Gene rfbS is present in both groups and encodes paratose synthase, which carries out a step parallel to that of abequose synthase, but the product is CDP-paratose. The DNA and inferred amino acid sequences are compared with those of rfbJ. We conclude that the genes are homologous, but the divergence is extremely ancient. Gene rfbE encodes CDP-tyvelose epimerase, which converts CDP-paratose to CDP-tyvelose in group D strains; the gene is active in group D strains, and we find it to be present in a mutant form in group A strains. These two genes encode the steps unique to groups A and D and, like rfbJ of group B, are of low G+C content, suggesting transfer from outside of salmonellae. The evolutionary origin of these genes is discussed.
Methylation of GATC sites is required for precise timing between rounds of DNA replication in Escherichia coli
Authors: Bakker A , Smith D W
Published: November 2016

We have used the Koppes and Nordstrøm (Cell 44:117-124, 1986) CsCl density transfer approach for analysis of DNA from exponentially growing, isogenic Escherichia coli dam+ and dam mutant cells to show that timing between DNA replication initiation events is precise in the dam+ cells but is essentially random in the dam cells. Thus, methylation of one or more GATC sites, such as those found in unusual abundance within the origin, oriC, is required for precise timing between rounds of DNA replication, and precise timing between initiation events is not required for cell viability. Both the dam-3 point mutant and the delta(dam)100 complete deletion mutant were examined. The results were independent of the mismatch repair system; E. coli mutH cells showed precise timing, whereas timing in the isogenic E. coli mutH delta(dam)100 double mutant was random. The mechanism is thus different from the role of Dam methylation in mismatch repair and probably involves conversion of hemimethylated GATC sites present in daughter origins just after initiation to a fully methylated state.
N alpha acetylation is required for normal growth and mating of Saccharomyces cerevisiae
Authors: Lee F J , Lin L W , Smith J A
Published: November 2016

Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eucaryotic proteins and is catalyzed by N alpha-acetyltransferase. The yeast enzyme is encoded by the AAA1 (amino-terminal alpha-amino acetyltransferase) gene. A null mutation (aaa1-1) created by gene replacement, while not lethal, slows cell growth and results in heterogeneous colony morphology. In comparison with wild-type cells, aaa1-1/aaa1-1 diploids cannot enter stationary phase, are sporulation defective, and are sensitive to heat shock. In addition, the aaa1-1 mutation specifically reduces mating functions of MATa cells. These results indicate that N alpha acetylation plays a crucial role in yeast cell growth and mating.
Mechanism of erythromycin-induced ermC mRNA stability in Bacillus subtilis
Authors: Bechhofer D H , Zen K H
Published: November 2016

In Bacillus subtilis, the ermC gene encodes an mRNA that is unusually stable (40-min half-life) in the presence of erythromycin, an inducer of ermC gene expression. A requirement for this induced mRNA stability is a ribosome stalled in the ermC leader region. This property of ermC mRNA was used to study the decay of mRNA in B. subtilis. Using constructs in which the ribosome stall site was internal rather than at the 5' end of the message, we show that ribosome stalling provides stability to sequences downstream but not upstream of the ribosome stall site. Our results indicate that ermC mRNA is degraded by a ribonucleolytic activity that begins at the 5' end and degrades the message in a 5'-to-3' direction.
Photosynthetic CO2 fixation and ribulose bisphosphate carboxylase/oxygenase activity of Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus
Authors: Steinberg N A , Meeks J C
Published: November 2016

The cyanobacterium Nostoc sp. strain UCD 7801, immediately after separation from pure cultures of a reconstituted symbiotic association with the bryophyte Anthoceros punctatus, exhibited a rate of light-dependent CO2 fixation that was eightfold lower than that measured in the free-living growth state. Ribulose bisphosphate carboxylase/oxygenase (RuBPC/O) specific activity was also eightfold lower in cell extracts of symbiotic strain 7801 relative to that in free-living cultures. The in vitro activity from symbiotic strain 7801 could not be increased by incubation under the standard RuBPC/O activation conditions. Polyclonal antibodies against the RuBPC/O large subunit were used in an enzyme-linked immunosorbent assay to determine that RuBPC/O accounted for 4.3 and 5.2% of the total protein in cell extracts of strain 7801 grown in symbiotic and free-living states, respectively. The results imply that the regulation of RuBPC/O activity in the symbiotic growth state is by a posttranslational mechanism rather than by an alteration in RuBPC/O protein synthesis. The amount of carboxyarabinitol bisphosphate required to irreversibly inhibit RuBPC/O activity of sybiotic cell extracts was 80% of that required for extracts of free-living cultures. This result indicates that any covalent modification of RuBPC/O in symbiotically associated Nostoc cells did not interfere with the ribulose bisphosphate binding site, since inactive enzyme also bound carboxyarabinitol bisphosphate.
Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO
Authors: Hoshino T , Kose K
Published: November 2016

The gene for the leucine-, isoleucine-, and valine-binding protein (LIVAT-BP) in Pseudomonas aeruginosa PAO was isolated, and its nucleotide sequence was determined. The gene consisted of 1,119 nucleotides specifying a protein of 373 amino acid residues. Determination of the N-terminal amino acid sequence of the LIVAT-BP purified from P. aeruginosa shock fluid suggested that the N-terminal 26 residues of the gene product are cleaved off posttranslationally, showing the characteristic features of procaryotic signal peptides. The amino acid composition of the mature product predicted from the nucleotide sequence was in good agreement with that of the purified LIVAT-BP. The plasmid carrying the LIVAT-BP gene restored the activity of the high-affinity branched-chain amino acid transport system (the leucine, isoleucine, valine [LIV-I] transport system) in the braC310 mutant of P. aeruginosa, confirming that braC is the structural gene for LIVAT-BP. The mutant LIVAT-BP lacking a 16-amino-acid peptide in the middle was found to be functional in the LIV-I transport system. LIVAT-BP showed extensive homology (51% identical) to the LIV- and leucine-specific-binding proteins of Escherichia coli K-12, which are coded for by the livJ and livK genes, respectively, suggesting that the role of the proteins in the LIV-I transport systems is analogous in both organisms.
Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli
Authors: Miller J F , Roy C R , Falkow S
Published: November 2016

The virulence regulon of Bordetella pertussis includes a trans-acting regulatory locus, bvg, that is required for expression of several virulence factors. The virulence control system also responds to environmental signals. We have reconstructed a bvg-dependent regulatory system in Escherichia coli by using bacteriophage lambda vectors carrying transcriptional fusions to lacZYA. Single-copy lacZYA fusions to the B. pertussis fhaB locus, which encodes the attachment factor filamentous hemagglutinin, were activated nearly 400-fold by pBR322 replicons carrying sequences that included bvg. In contrast, bvg had no effect on the pertussis toxin operon (ptxA-E) promoter in E. coli as measured by ptxA-lacZ expression. Environmental signals that modulate expression of virulence genes in B. pertussis had a pronounced effect on bvg-mediated activation of fhaB-lacZ. MgSO4, nicotinic acid, and low temperature resulted in decreases in beta-galactosidase activities of 175-, 115-, and 45-fold respectively. Sensory transduction and transcriptional activation were tightly coupled, and both required an intact bvg locus as determined by 5' and 3' deletions that eliminated both activities.
rpoZ, encoding the omega subunit of Escherichia coli RNA polymerase, is in the same operon as spoT
Authors: Gentry D R , Burgess R R
Published: November 2016

Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega. This subunit consists of 91 amino acids with a molecular weight of 10,105. We previously reported the cloning and sequencing of the gene encoding omega, which we call rpoZ (D. R. Gentry and R. R. Burgess, Gene 48:33-40, 1986). We constructed an rpoZ insertion mutation by placing a kanamycin resistance cassette into the coding region of the rpoZ gene. Purified RNA polymerase from strains carrying this mutation lacked detectable omega. We found that the insertion mutation conferred a slow-growth phenotype when introduced into most strains. We mapped the position of rpoZ on the E. coli chromosome by genetic techniques and by examining the restriction map of the whole chromosome and found that rpoZ maps around 82 min, very close to spoT. We determined that the slow-growth phenotype of the insertion mutant is suppressed in relA mutants and that the rpoZ insertion results in a classical SpoT- phenotype. This finding strongly suggests that rpoZ is upstream of spoT in the same operon and that the slow-growth phenotype elicited by the insertion mutation is due to polarity on spoT.