bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigenic mosaic of Methanogenium spp.: analysis with poly- and monoclonal antibody probes
Authors: Macario A J , Dugan C B , de Macario E C
Published: November 2016

Eight well-characterized Methanogenium strains, including the six described type strains, were analyzed with poly- and monoclonal antibody probes to examine the antigenic mosaic of the genus. The pattern of cross-reactions showed that the mosaic is complex and varies with the strains; thus, these organisms have developed a considerable antigenic diversity, which is expressed in their envelopes. Every strain shared at least one determinant with at least one other strain, demonstrating the antigenic cohesiveness of the group. This finding, together with the fact that most strains displayed a distinctive antigenic fingerprint (notwithstanding the limited number of probes available), emphasizes the potential of antibodies for rapid identification of new isolates and for direct elucidation of Methanogenium strains in microbial mixtures.
DNA sequence and in vitro expression of the B875 light-harvesting polypeptides of Rhodobacter sphaeroides
Authors: Kiley P J , Donohue T J , Havelka W A , Kaplan S
Published: November 2016

The genes for the Rhodobacter sphaeroides light-harvesting B875-beta, and B875-alpha polypeptides (pufB and pufA) are closely linked to the genes for the reaction center L and reaction center M polypeptides (pufL and pufM) on what has been termed the puf operon (gene order, pufB, A, L, M). The DNA sequence of the pufB and pufA structural genes from wild-type R. sphaeroides 2.4.1 was determined and aligned with the available amino acid sequence of the wild-type B875-beta and B875-alpha polypeptides. The relative levels of the B875-beta and B875-alpha and the reaction center L and reaction center M polypeptides synthesized in a homologous cell-free transcription-translation system were compared with those found in vivo. Analysis of the gene products produced in vitro with plasmids containing deletions upstream of the pufB structural gene identified a region of DNA required for expression of the B875-beta and B875-alpha polypeptides. These results support the hypothesis that the mapped 5' termini of the large and small puf operon transcripts represent transcription initiation sites.
Structure of the Azotobacter vinelandii surface layer
Authors: Bingle W H , Whippey P W , Doran J L , Murray R G , Page W J
Published: November 2016

Electron microscopy of the Azotobacter vinelandii tetragonal surface array, negatively stained with ammonium molybdate in the presence of 1 mM calcium chloride, showed an apparent repeat frequency of 12 to 13 nm. Image processing showed dominant tetrad units alternating with low-contrast cruciform structures formed at the junction of slender linkers extending from corner macromolecules of four adjoining dominant units. The actual unit cell showed p4 symmetry, and a = b = 18.4 nm. Distilled water extraction of the surface array released a multimeric form of the single 60,000 molecular-weight protein (S protein) which constitutes the surface layer. The molecular weight of the multimer was estimated at 255,000 by gel filtration, indicating a tetrameric structure of four identical subunits and suggesting that this multimer was the morphological subunit of the S layer. Tetrameric S protein exhibited low intrinsic stability once released from the outer membrane, dissociating into monomers when incubated in a variety of buffers including those which served as the base for defined media used to cultivate A. vinelandii. The tetramer could not be stabilized in these buffers at any temperature between 4 and 30 degrees C, but the addition of 2 to 5 mM Ca2+ or Mg2+ completely prevented its dissociation into monomers. Circular dichroism measurements indicated that the secondary structure of the tetramer was dominated by aperiodic and beta-sheet conformations, and the addition of Ca2+ did not produce any gross changes in this structure. Only the tetrameric form of S protein was able to reassemble in vitro in the presence of divalent cations onto the surface of cells stripped of their native S layer.
Acetohydroxy acid synthase I is required for isoleucine and valine biosynthesis by Salmonella typhimurium LT2 during growth on acetate or long-chain fatty acids
Authors: Dailey F E , Cronan J E , Maloy S R
Published: November 2016

Salmonella typhimurium LT2 normally expresses two acetohydroxy acid synthases (AHAS I and AHAS II). The function of AHAS I in this organism was unclear, since AHAS I-deficient (ilvBN) mutants of LT2 grew well on glucose or succinate minimal media, whereas AHAS II-deficient (ilvGM) mutants requried isoleucine for normal growth on glucose minimal media. We report that AHAS I-deficient mutants of S. typhimurium required isoleucine and valine for growth on acetate or oleate minimal media, whereas AHAS II-deficient mutants were able to grow on these media without isoleucine supplementation.
Purification and use of Methanobacterium wolfei pseudomurein endopeptidase for lysis of Methanobacterium thermoautotrophicum
Authors: Kiener A , König H , Winter J , Leisinger T
Published: November 2016

The pseudomurein-degrading enzyme from autolysates of Methanobacterium wolfei was purified approximately 500-fold to electrophoretic homogeneity by ion-exchange chromatography under anaerobic conditions. Analysis of the soluble cell wall fragments produced by the pure enzyme from a cell wall preparation of M. thermoautotrophicum indicated that it is a peptidase hydrolyzing the epsilon-Ala-Lys bond of pseudomurein. A partially purified preparation of pseudomurein endopeptidase was free of nuclease activity and thus proved useful for the preparation in high yields of undegraded chromosomal and plasmid DNA from M. thermoautotrophicum. The partially purified enzyme was also used for the preparation of protoplasts, which were stabilized by 0.8 M sucrose. Under growth conditions the protoplasts produced methane and increased up to 100-fold in size, but failed to regenerate a cell wall.
Nitrogen starvation mediated by DL-7-azatryptophan in the cyanobacterium Anabaena sp. strain CA
Authors: Chen C H , Van Baalen C , Tabita F R
Published: November 2016

The addition of DL-7-azatryptophan (AZAT), a tryptophan analog, to continuous cultures of Anabaena sp. strain CA grown with 10 mM nitrate as the nitrogen source resulted in the differentiation of heterocysts. Analysis of the intracellular amino acid pools of Anabaena sp. strain CA after the addition of AZAT showed a marked decline in the intracellular glutamate pool and a slight increase in the levels of glutamine. The in vitro activity of glutamate synthase, the second enzyme involved in primary ammonia assimilation in Anabaena spp., was partially inhibited by the presence of AZAT at concentrations which are effective in triggering heterocyst formation (15% inhibition at 10 microM AZAT and up to 85% inhibition at 1.0 mM AZAT). Azaserine, a glutamine analog and potent glutamate synthase inhibitor, had no effect on the triggering of heterocyst development from undifferentiated batch and continuous cultures of Anabaena sp. strain CA. However, the presence of 1.0 microM azaserine significantly decreased the intracellular glutamate pool and increased the glutamine pool. The addition of AZAT also caused a decrease in the C-phycocyanin content of Anabaena sp. strain CA as a result of its proteolytic degradation. AZAT also had an inhibitory effect on the nitrogenase activity of Anabaena sp. strain CA. All these results suggest that AZAT causes a general nitrogen starvation of Anabaena sp. strain CA filaments, triggering heterocyst synthesis.
Effects of Rhizobium meliloti nif and fix mutants on alfalfa root nodule development
Authors: Hirsch A M , Smith C A
Published: November 2016

Ineffective alfalfa nodules were examined at the light and electron microscope level after inoculation with Rhizobium meliloti strains with mutations in nif and fix genes. All the mutant strains induced nodules that contained elongated bacteroids within the host cells, but the bacteroids quickly senesced. The nodules were small and numerous, and the host cells also exhibited symptoms of an ineffective symbiosis. nifB, fixA, and fixB bacteroids appeared to be completely differentiated (by ultrastructural criteria), i.e., as bacteroids developed, they increased in diameter and length and their cytoplasm underwent a change from homogeneous and electron dense to heterogeneous and electron transparent after enlargement. In contrast, nifA bacteroids rarely matured to this state. The bacteroids degenerated at an earlier stage of development and did not become electron transparent.
Cell cycle changes in the buoyant density of exponential-phase cells of Streptococcus faecium
Authors: Dicker D T , Higgins M L
Published: November 2016

Cell buoyant densities were determined by centrifugation in Percoll gradients containing exponential-phase cells of Streptococcus faecium ATCC 9790 grown at a mass doubling time of about 33 min. This bacterium showed the highest average density values (1.13 g/ml) measured to date for any eucaryotic or procaryotic organism. Fractions having the highest densities were enriched with cells that were in the process of dividing or had just divided. These high-density fractions were also enriched with cells that had newly initiated sites of cell wall growth. It appears that S. faecium shows minimum cell densities in the midportion of its cycle.
In vitro expression of a Tn9-derived chloramphenicol acetyltransferase gene fusion by using a Bacillus subtilis system
Authors: Zaghloul T I , Doi R H
Published: November 2016

A coupled in vitro protein-synthesizing system has been developed with components derived totally from Bacillus subtilis. The system synthesized specific gene products from various exogenous DNA templates, including B. subtilis phage phi 29, plasmid pUB110, and a heterologous B. subtilis-Escherichia coli gene fusion containing the transposon Tn9-derived chloramphenicol acetyltransferase (cat) gene. The gene fusion product was able to show CAT activity, bind specifically to a Sephacryl-chloramphenicol column, and react immunologically against anti-CAT antiserum. The fidelity of this in vitro system was demonstrated by the synthesis of gene products identical to that made in vivo. We suggest that this system may be used to study the regulation of gene expression in vitro.
secD, a new gene involved in protein export in Escherichia coli
Authors: Gardel C , Benson S , Hunt J , Michaelis S , Beckwith J
Published: November 2016

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.
Occurrence in Bacillus megaterium of uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine, intermediates in the biosynthesis of galactosamine-6-phosphate polymer
Authors: Nishikawa J , Tada M , Takubo Y , Nishihara T , Kondo M
Published: November 2016

We isolated two galactosamine derivatives from Bacillus megaterium sporulating cells by lectin affinity chromatography followed by DEAE-Sephadex A-25 chromatography. From chemical analyses and measurements of these compounds, it was determined that one was uridine 5'-diphospho-N-acetylgalactosamine and that the other was uridine 5'-diphosphogalactosamine. They appeared in the middle stage of sporulation and disappeared during the period when galactosamine-6-phosphate is deposited on the forespore surface. These results suggest that uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine are intermediates in the biosynthesis of the galactosamine-6-phosphate polymer, a backbone structure of the exosporium.
Evolutionary origin of pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1
Authors: Yamamoto T , Gojobori T , Yokota T
Published: November 2016

Three families of the evolutionarily related pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1, a family of cholera enterotoxin (CT) and heat-labile enterotoxin (LT) including CT, LTh, and LTp, a family of heat-stable enterotoxin I (STI) including STIa and STIb, and a family of K88 enteroadhesion fimbriae including K88ab, K88ac, and K88ad were analyzed for synonymous (silent) nucleotide substitutions by using the gene nucleotide sequences of earlier reports and the LTp gene nucleotide sequence presented in this paper. The data suggested that the divergences between LT and CT and between STIa and STIb occurred in the remote past, whereas those between LTh and LTp and between members of the K88 family occurred very recently. We concluded that the LT gene is a foreign gene that has been acquired by E. coli to form an enteropathogen. This provides evolutionary evidence of species-to-species transfer of pathogenic determinants in procaryotes.
Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum
Authors: Hartmann A , Burris R H
Published: November 2016

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.
Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis
Authors: Torres-Cabassa A S , Gottesman S
Published: November 2016

lon mutants of Escherichia coli K-12 are defective in an ATP-dependent protease, are UV sensitive, and overproduce the capsular polysaccharide colanic acid. Six structural genes needed for capsular polysaccharide synthesis (cps) are transcriptionally regulated by lon as well as by three other regulatory genes, rcsA, -B, and -C (S. Gottesman, P. Trisler, and A. S. Torres-Cabassa, J. Bacteriol. 162:1111-1119, 1985). We have cloned rcsA, the gene for a positive regulator of capsule synthesis, onto multicopy plasmids and defined the gene by both insertions and deletions. The product of rcsA has been identified as an unstable protein of 27 kilodaltons. RcsA has a half-life of 5 min in lon+ cells and one of 20 min in lon cells. The availability of RcsA is the limiting factor for capsule synthesis; doubling the gene dosage of rcsA+ significantly increases expression of cps genes. Our results are consistent with a model in which the presence of a lon mutation increases the synthesis of capsular polysaccharide via stabilization of RcsA.
Characterization of a membrane-associated serine protease in Escherichia coli
Authors: Palmer S M , St John A C
Published: November 2016

Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively (M. Pacaud, J. Bacteriol. 149:6-14, 1982). The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. We termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [3H]diisopropylfluorophosphate as an active-site labeling reagent, we determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, and endogenous, periplasmic inhibitor of trypsin.
The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export
Authors: Homma M , Komeda Y , Iino T , Macnab R M
Published: November 2016

flaFIX, the structural gene for the periplasmic P ring of the flagellar basal body of Salmonella typhimurium, was cloned. Two gene products with apparent molecular weights of 38,000 and 40,000 were identified by minicell analysis. Data from pulse-chase and membrane fractionation experiments and data on the inhibitory effect of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone all indicated that the 40-kilodalton protein was a precursor form which, after export across the cytoplasmic membrane accompanied by cleavage of a signal peptide, gave rise to the mature protein in the periplasm. The N-terminal amino acid sequence of the FlaFIX protein, predicted from the DNA sequence, conformed well to known signal peptide sequences. The results indicate that the P-ring protein of the basal body (unlike flagellin and possible some other external flagellar components) crosses the cytoplasmic membrane in a conventional signal peptide-dependent manner.
Characterization of the virE locus of Agrobacterium tumefaciens plasmid pTiC58
Authors: Hirooka T , Rogowsky P M , Kado C I
Published: November 2016

The virE locus that is responsible for the efficiency of infection by Agrobacterium tumefaciens (T. Hirooka and C. Kado, J. Bacteriol. 168:237-243, 1986) is located next to the right boundary of the virulence (Vir) region of the nopaline plasmid pTiC58. This locus is very similar to the virE locus of octopine type Ti plasmids on the basis of nucleotide and amino acid sequence comparisons as well as genetic complementation analyses. The nucleotide sequence of virE revealed three open reading frames, arranged as an operon, with a potential coding capacity for proteins of 9, 7.1, and 63.5 kilodaltons. The promoter region of virE was analyzed by using gene fusions to promoterless cat and lux genes. Two different promoters were detected, one which operates in A. tumefaciens and one which operates in Escherichia coli. virE is transcribed from left to right toward the T region. In A. tumefaciens, the expression of virE was induced by acetosyringone and required the presence of pTiC58.
Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase
Authors: Boquet P L , Manoil C , Beckwith J
Published: November 2016

The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques. The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions. For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export. The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.
Specific magnesium-dependent diadenosine 5',5'''-P1,P3-triphosphate pyrophosphohydrolase in Escherichia coli
Authors: Hurtado C , Ruíz A , Sillero A , Sillero M A
Published: November 2016

A specific Mg2+-dependent bis(5'-adenosyl)-triphosphatase (EC 3.6.1.29) was purified 270-fold from Escherichia coli. The enzyme had a strict requirement for Mg2+. Other divalent cations, such as Mn2+, Ca2+, or Co2+, were not effective. The products of the reaction with bis(5'-adenosyl) triphosphate (Ap3A) as the substrate were ADP and AMP in stoichiometric amounts. The Km for Ap3A was 12 +/- 5 microM. Bis(5'-adenosyl) di-, tetra-, and pentaphosphates, NAD+, ATP, ADP, AMP, glucose 6-phosphate, p-nitrophenylphosphate, bis-p-nitrophenylphospate, and deoxyribosylthymine-5'-(4-nitrophenylphosphate) were not substrates of the reaction. The enzyme had a molecular mass of 36 kilodaltons (as determined both by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of 4.84 +/- 0.05, and a pH optimum of 8.2 to 8.5. Zn2+, a known potent inhibitor of rat liver bis(5'-adenosyl)-triphosphatase and bis(5'-guanosyl)-tetraphosphatase (EC 3.6 1.17), was without effect. The enzyme differs from the E. coli diadenosine 5',5'''-P1, P4-tetraphosphate pyrophosphohydrolase which, in the presence of Mn2+, also hydrolyzes Ap3A.
Suppression of a signal sequence mutation by an amino acid substitution in the mature portion of the maltose-binding protein
Authors: Cover W H , Ryan J P , Bassford P J , Walsh K A , Bollinger J , Randall L L
Published: November 2016

An unusual spontaneous pseudorevertant of an Escherichia coli strain carrying the signal sequence point mutation malE14-1 was characterized. The suppressor mutation, malE2261, resulted in a single substitution of an aspartyl residue for a tyrosyl residue at position 283 in the sequence of the mature maltose-binding protein. The precursor retained the malE14-1 point mutation in the signal sequence. The pseudorevertant carrying both malE14-1 and malE2261 exported twice the amount of maltose-binding protein as that of the mutant carrying the malE14-1 allele alone but only 18% of the amount exported by a strain producing wild-type maltose-binding protein. A strain carrying the suppressor allele malE2261 in combination with a wild-type signal sequence exported normal quantities of maltose-binding protein to the periplasm. Mature MalE2261 had a Kd for maltose of 27 microM, compared with 3.6 microM for mature wild-type maltose-binding protein. The precursor species than contained both changes resulting from malE14-1 and malE2261 was significantly less stable in the cytoplasm than was the precursor containing only the change encoded by malE14-1.
New phenotypes associated with mucAB: alteration of a MucA sequence homologous to the LexA cleavage site
Authors: Marsh L , Walker G C
Published: November 2016

Most mutagenesis by UV and many chemicals in Escherichia coli requires the products of the umuDC operon or an analogous plasmid-derived operon mucAB. Activated RecA protein is also required for, or enhances, this process. MucA and UmuD proteins share homology with the LexA protein, suggesting that they might interact with the RecA protein as LexA does. We used oligonucleotide-directed mutagenesis to alter a site in MucA homologous to the Ala-Gly cleavage site of LexA. The mutation, termed mucA101(Glu26), results in a change of Gly26 of MucA to Glu26. A lexA(Def) recA441 umuC122::Tn5 strain carrying a mucA101(Glu26)B+ plasmid did not exhibit the greatly increased frequency of spontaneous mutagenesis in response to RecA activation that a strain carrying a mucA+B+ plasmid did but retained a basal recA-dependent ability to confer increased spontaneous mutagenesis that was independent of the state of RecA activation. These results are consistent with a model in which RecA plays two distinct roles in mutagenesis apart from its role in the cleavage of LexA. A pBR322-derived plasmid carrying mucA+B+, but not one carrying mucA101(Glu26)B+, inhibited the UV induction of SOS genes, suggesting that MucA+ and MucA(Glu26) proteins may have different abilities to compete with LexA for activated RecA protein. The spectrum of UV-induced mutagenesis was also altered in strains carrying the mucA101(Glu26) mutation. These results are consistent with the hypothesis that activated RecA protein interacts with wild-type MucA protein, possibly promoting proteolytic cleavage, and that this interaction is responsible for facilitating certain mutagenic processes.
Nucleotide sequence of the streptothricin acetyltransferase gene from Streptomyces lavendulae and its expression in heterologous hosts
Authors: Horinouchi S , Furuya K , Nishiyama M , Suzuki H , Beppu T
Published: November 2016

The nucleotide sequence of the streptothricin acetyltransferase (STAT) gene from streptothricin-producing Streptomyces lavendulae predicts a 189-amino-acid protein of molecular weight 20,000, which is consistent with that determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme. The amino acid composition and the NH2-terminal sequence determined by using the purified protein are in good agreement with those predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. High-resolution S1 nuclease protection mapping suggests that transcription initiates at or near the adenine residue which is the first position of the translational initiation triplet (AUG) of STAT. Another open reading frame located just upstream of the STAT gene was detected and contains a region bearing a strong resemblance to DNA-binding domains which are conserved in known DNA-binding proteins. By addition of promoter signals and a synthetic ribosome-binding (Shine-Dalgarno) sequence at an appropriate position upstream of the STAT translational start codon, the STAT gene confers streptothricin resistance on Escherichia coli and Bacillus subtilis. The STAT coding sequence with both the promoter of a B. subtilis cellulase gene and a synthetic Shine-Dalgarno sequence was functionally expressed in Streptomyces lividans, which suggests that the addition of an artificial leader upstream of the translational initiation codon (AUG) does not significantly influence the translation of STAT.
Self-protection of Pseudomonas syringae pv. "tabaci" from its toxin, tabtoxinine-beta-lactam
Authors: Knight T J , Durbin R D , Langston-Unkefer P J
Published: November 2016

An extracellular toxin, tabtoxinine-beta-lactam (T beta L), is produced by Pseudomonas syringae pv. "tabaci." This toxin irreversibly inhibits its target, glutamine synthetase; yet P. syringae pv. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection of P. syringae pv. "tabaci," we compared the effects of T beta L on Tox+ (T beta L-producing, insensitive to T beta L) and Tox- (T beta L nonproducing, sensitive to T beta L) strains. The extent of protection afforded to the Tox- strain when induced to adenylylate glutamine synthetase was tested. We concluded that an additional protection mechanism was required. A detoxification activity was found in the Tox+ strain which opens the beta-lactam ring of T beta L to produce the inactive, open-chain form, tabtoxinine. Whole cells of the Tox+ strain incubated for 24 h with [14C]T beta L (0.276 mumol/3 X 10(10) cells) contained [14C]tabtoxinine (0.056 mumol), and the medium contained T beta L (0.226 mumol). Extracts of spheroplasts of the Tox+ stain also converted T beta L to tabtoxinine, whereas extracts of the Tox- strain did not alter T beta L. The conversion was time dependent and stoichiometric and was destroyed by boiling for 30 min or by the addition of 5 mM EDTA. Penicillin, a possible substrate and competitive inhibitor of this lactamase activity, inhibited the conversion of T beta L to tabtoxinine. Periplasmic fluid did not catalyze the conversion of T beta L.
Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO
Authors: Kropinski A M , Lewis V , Berry D
Published: November 2016

Growth of Pseudomonas aeruginosa PAO1 at 15 to 45 degrees C in tryptic soy broth resulted in changes in the lipids, lipopolysaccharides (LPSs), and outer membrane proteins of the cells. Cells grown at 15 degrees C contained, relative to those cultivated at 45 degrees C, increased levels of the phospholipid fatty acids hexadecenoate and octadecenoate and reduced levels of the corresponding saturated fatty acids. Furthermore, the lipid A fatty acids also showed thermoadaptation with decreases in dodecanoic and hexadecanoic acids and increases in the level of 3-hydroxydecanoate and 2-hydroxdodecanoate as the growth temperature decreased. In addition, LPS extracted from cells cultivated at the lower temperatures contained a higher content of long-chain S-form molecules than that isolated from cells grown at higher temperatures. On the other hand, the percentage of LPS cores substituted with side-chain material decreased from 37.6 mol% at 45 degrees C to 19.3 mol% at 15 degrees C. The outer membrane protein profiles indicated that at low growth temperatures there was an increase in a polypeptide with an apparent molecular weight of 43,000 and decreases in the content of 21,000 (protein H1)- and 27,500-molecular-weight proteins.
Activities of oxidative enzymes in mycoplasmas
Authors: Constantopoulos G , McGarrity G J
Published: November 2016

The activities of several oxidoreductases were measured in three fermentative and two nonfermentative Mycoplasma species that were grown under aerobic or anaerobic conditions. Acholeplasma laidlawii MG, Mycoplasma hyorhinis GDL, and Mycoplasma pneumoniae FH had very high apparent activities of pyruvate dehydrogenase and pyruvate dehydrogenase complex compared with the activities of mammalian fibroblasts or human platelet-enriched preparations, while Mycoplasma salivarium VV and Mycoplasma arthritidis 07 had very low apparent activities of these two enzymes. Strictly anaerobic growth diminished both enzymatic activities. The activity of alpha-ketoglutarate dehydrogenase complex was minimal in all five mycoplasmas that were grown under aerobic conditions, anaerobic conditions, or both. All the mycoplasmas that were examined exhibited lactate dehydrogenase and NADH-dichlorophenol indophenol oxidoreductase activities. The properties of mycoplasmal pyruvate dehydrogenase complex suggest that it differs from the mammalian enzyme.
Purification and properties of a protein linked to the soluble hydrogenase of hydrogen-oxidizing bacteria
Authors: Kärst U , Suetin S , Friedrich C G
Published: November 2016

In Alcaligenes eutrophus, the formation of the hydrogenases and of five new peptides is subject to the hydrogenase control system. Of these, the B peptide was purified to homogeneity. This protein (Mr, 37,500) was composed of two identical subunits (Mr, 18,800). Antibodies against the B protein were used for its quantification by rocket immunoelectrophoresis. About 4% of the total protein consisted of the B protein; its molar ratio to the NAD-linked hydrogenase was about 4:1. The B protein appeared to be associated with the NAD-linked hydrogenase, as shown by gel filtration analysis with Sephadex G-200. The B protein was not detected in cells that had not expressed the hydrogenase proteins or that lacked the genetic information of the hydrogen-oxidizing character; it was also not detected in Tn5 insertional mutants that were unable to form soluble hydrogenase antigens. Immunochemical analysis of other species and genera than A. eutrophus revealed that only strains able to form a NAD-linked hydrogenase also formed B-protein antigens. The B protein is not required for the catalytic activity of soluble hydrogenase in vitro; its function is at present unknown.
Campylobacter pylori, the spiral bacterium associated with human gastritis, is not a true Campylobacter sp
Authors: Romaniuk P J , Zoltowska B , Trust T J , Lane D J , Olsen G J , Pace N R , Stahl D A
Published: November 2016

Comparison of partial 16S rRNA sequences from representative Campylobacter species indicates that the Campylobacter species form a previously undescribed basic eubacterial group, which is related to the other major groups only by very deep branching. This analysis was extended to include the spiral bacterium associated with human gastritis, Campylobacter pylori (formerly Campylobacter pyloridis). The distance between C. pylori and the other Campylobacter species is sufficient to exclude the pyloric organism from the Campylobacter genus. The results indicate that C. pylori is more closely related to Wolinella succinogenes than it is to the other Campylobacter species inspected. Another close relative of the campylobacters was found to be Thiovulum, a sulfide-dependent marine bacterium.
Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus spp
Authors: Carmi O A , Stewart G S , Ulitzur S , Kuhn J
Published: November 2016

We report the construction and use of a new promoter probe vehicle capable of allowing extremely sensitive measurements of transcriptional activity promoted from random, chromosomal DNA fragment inserts. Coupled with the advantage of sensitivity, the detection system is noninvasive, nondestructive, and provides real-time reportage of expression potential. These latter aspects make it an especially valuable system for a continuing analysis of the complex transcriptional regulation patterns now recognized as a dominant control feature during the differentiation and morphogenesis characteristic of the sporulation cycle in Bacillus species. In this respect we describe the isolation of DNA fragments from B. megaterium and B. subtilis capable of initiating transcription in both the respective parent organisms and, in certain instances, also in Escherichia coli. Detailed luminescence studies showed that several promoter regions which are entirely or substantially developmentally controlled were isolated.
Outer membrane protein mediating iron uptake via pyoverdinpss, the fluorescent siderophore produced by Pseudomonas syringae pv. syringae
Authors: Cody Y S , Gross D C
Published: November 2016

In an iron-limited environment Pseudomonas syringae pv. syringae B301D produces a yellow-green fluorescent siderophore called pyoverdinpss which functions in high-affinity iron transport. Two-dimensional electrophoretic comparisons of the outer membrane proteins of strain B301D identified nine proteins which were expressed at low (50 nM) but not at high (10 microM) iron concentrations. Except for the minor protein 8e, the iron-regulated proteins exhibited high molecular weights ranging from approximately 74,000 to 80,000. A mutant of strain B301D incapable of iron uptake (Iu-) from ferric pyoverdinpss lacked the 74,000-molecular-weight protein 4a, which was the major iron-regulated outer membrane protein. In contrast, a nonfluorescent mutant (Flu-) unable to synthesize pyoverdinpss showed no quantitative or qualitative difference in its outer membrane profile from that of the wild-type strain. In plant pathogenicity tests the Iu- and Flu- strains caused typical brown necrotic and sunken lesions in immature sweet cherry fruit which were indistinguishable from those of the wild-type strain. Thus, excretion of pyoverdinpss and subsequent Fe(III) uptake do not have a determinative role in the pathogenicity or virulence of P. syringae pv. syringae.
A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus
Authors: Renalier M H , Batut J , Ghai J , Terzaghi B , Gherardi M , David M , Garnerone A M , Vasse J , Truchet G , Huguet T
Published: November 2016

A 290-kilobase (kb) region of the Rhizobium meliloti 2011 pSym megaplasmid, which contains nodulation genes (nod) as well as genes involved in nitrogen fixation (nif and fix), was shown to carry at least six sequences repeated elsewhere in the genome. One of these reiterated sequences, about 5 kb in size, had previously been identified as part of a cluster of fix genes located 220 kb downstream of the nifHDK promoter. Deletion of the reiterated part of this fix cluster does not alter the symbiotic phenotype. Deletion of the second copy of this reiterated sequence, which maps on pSym 40 kb upstream of the nifHDK promoter, also has no effect. Deletion of both of these copies however leads to a Fix- phenotype, indicating that both sequences carry functionally reiterated fix gene(s). The fix copy 40 kb upstream of nifHDK is part of a symbiotic cluster which also carries a nod locus, the deletion of which produces a marked delay in nodulation.
Peroxisomal localization and activation by bivalent metal ions of ureidoglycolate lyase, the enzyme involved in urate degradation in Candida tropicalis
Authors: Takada Y , Tsukiji N
Published: November 2016

Ureiodoglycolate lyase (EC 4.3.2.3) was found only in the peroxisomes in urate-induced Candida tropicalis. The enzyme was markedly activated by the bivalent metal ions Mn2+, Fe2+, and Ni2+. The activation by Mn2+ was suggested to be the result of its binding to the apoenzyme.
Streptococcus faecium mutants that are temperature sensitive for cell growth and show alterations in penicillin-binding proteins
Authors: Canepari P , Lleò M M , Fontana R , Satta G
Published: November 2016

The penicillin-binding proteins (PBPs) of 209 cell division (or growth) temperature-sensitive mutants of Streptococcus faecium were analyzed in this study. A total of nine strains showed either constitutive or temperature-sensitive conditional damage in the PBPs. Analysis of these nine strains yielded the following results: one carried a PBP 1 constitutively showing a lower molecular weight; one constitutively lacked PBP 2; two lacked PBP 3 at 42 degrees C, but not at 30 degrees C; one was normal at 30 degrees C but at 42 degrees C lacked PBP 3 and overproduced PBP 5; two were normal at 42 degrees C and lacked PBP 5 at 30 degrees C; one constitutively lacked PBP 5; and one carried a PBP 6 constitutively split in two bands. The mutant lacking PBP 3 and overproducing PBP 5 continued to grow at 42 degrees C for 150 min and then lysed. Revertants selected for growth capability at 42 degrees C from the mutants altered in PBPs 5 and 6 maintained the same PBP alterations, while those isolated from the strains with altered PBP 1 or lacking PBP 2 or PBP 3 showed a normal PBP pattern. Penicillin-resistant derivatives were isolated at 30 degrees C from the mutants lacking PBP 2 and from that lacking PBP 3. All these derivatives continued to show the same PBP damage as the parents, but overproduced PBP 5 and grew at 42 degrees C. These findings indicate that high-molecular-weight, but not low-molecular-weight, PBPs are essential for cell growth in S. faecium. This is in complete agreement with previous findings obtained with a different experimental system. On the basis of both previous and present data it is suggested that PBPs 1, 2, and 3 appear necessary for cell growth at optimal temperature (and at maximal rate), but not for cell growth at a submaximal one (or at a reduced rate), and an overproduced PBP 5 is capable of taking over the function of PBPs 1, 2, and 3.
Selection of mutations that alter the osmotic control of transcription of the Salmonella typhimurium proU operon
Authors: Druger-Liotta J , Prange V J , Overdier D G , Csonka L N
Published: November 2016

We isolated 60 independent mutations, designated osmX, in Salmonella typhimurium that result in constitutive expression of the normally osmoregulated proU operon. Each of the osmX mutations is closely linked to the proU locus and cis-dominant over the osmX+ allele in diploid strains. These results suggest that the mutations are probably in the 5' transcriptional control region of the proU operon. Our failure to obtain either recessive or unlinked mutations that altered the osmotic control of transcription of the proU operon suggests that transcriptional regulation of the gene is not under the negative control of a repressor protein that is dispensable for cell viability. We discuss possible models for the mechanism of osmotic regulation of transcription of the proU operon.
Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli
Authors: Kalyananda M K , Engel R , Tropp B E
Published: November 2016

When either 3H-labeled L-glyceraldehyde or 3H-labeled L-glyceraldehyde 3-phosphate (GAP) was added to cultures of Escherichia coli, the phosphoglycerides were labeled. More than 81% of the label appeared in the backbone of the phosphoglycerides. Chromatographic analyses of the labeled phosphoglycerides revealed that the label was normally distributed into phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. These results suggest that L-glyceraldehyde is phosphorylated and the resultant L-GAP is converted into sn-glycerol 3-phosphate (G3P) before being incorporated into the bacterial phosphoglycerides. Cell-free bacterial extracts catalyzed an NADPH-dependent reduction of L-GAP to sn-G3P. The partially purified enzyme was specific for L-GAP and recognized neither D-GAP nor dihydroxyacetone phosphate as a substrate. NADH could not replace NADPH as a coenzyme. The L-GAP:NADPH oxidoreductase had an apparent Km of 28 and 35 microM for L-GAP and NADPH, respectively. The enzyme was insensitive to sulfhydryl reagents and had a pH optimum of approximately 6.6. The phosphonic acid analog of GAP, 3-hydroxy-4-oxobutyl-1-phosphonate, was a substrate for the reductase, with an apparent Km of 280 microM.
Variability in molybdenum uptake activity in Bradyrhizobium japonicum strains
Authors: Graham L , Maier R J
Published: November 2016

Twenty naturally occurring strains of Bradyrhizobium japonicum in 11 serogroups were screened for the ability to take up Mo as bacteroids from soybean root nodules. The strains varied greatly in their ability to take up Mo in a 1-min period. The best strain was USDA 136, which had an Mo uptake activity of almost 3.0 pmol/min per mg of bacteroid (dry weight). In contrast, the poorest strain, USDA 62, had an Mo uptake activity of 0.35 pmol of Mo per min per mg of bacteroid. There were similarities in Mo uptake ability among most of the same serogroup members. The variability in Mo uptake rates between the best (USDA 136 and USDA 122) and poorest (USDA 62 and USDA 140) strains was attributed to their differing affinities for Mo. Double-reciprocal plots of velocity versus substrate indicated a Km for USDA 136 and USDA 122 of 0.045 and 0.054 microM, respectively, whereas strains USDA 62 and USDA 140 both exhibited an apparent Km for MoO42- of about 0.36 microM. The two strains with the higher-affinity Mo binding also accumulated four to five times as much Mo over a 30-min period as the other strains. Soybeans were grown in Mo-deficient and Mo-supplemented conditions after inoculation with the three top-ranking Mo uptake strains and the three poorest Mo uptake strains. Two separate greenhouse studies indicated that Mo supplementation significantly increased the N2 fixation activity of USDA 140 nodules; up to a 35% increase in specific nitrogen fixation activity of nodules due to Mo supplementation was observed. Strain USDA 62 nodule N2 fixation responded positively to Mo supplementation in one of the two experiments. The results indicate that MoO42- transport and, specifically, affinity for Mo by the bacteroid may ultimately affect symbiotic N2 fixation activity. Attempts to reactivate nitrogenase by adding molybdate to bacteroids from plants grown in Mo-deficient conditions were unsuccessful.
Periodic synthesis of phospholipids during the Caulobacter crescentus cell cycle
Authors: O'Neill E A , Bender R A
Published: November 2016

Net phospholipid synthesis is discontinuous during the Caulobacter crescentus cell cycle with synthesis restricted to two discrete periods. The first period of net phospholipid synthesis begins in the swarmer cell shortly after cell division and ends at about the time when DNA replication initiates. The second period of phospholipid synthesis begins at a time when DNA replication is about two-thirds complete and ends at about the same time that DNA replication terminates. Thus, considerable DNA replication, growth, and differentiation (stalk growth) occur in the absence of net phospholipid synthesis. In fact, when net phospholipid synthesis was inhibited by the antibiotic cerulenin through the entire cell cycle, both the initiation and the elongation phases of DNA synthesis occurred normally. An analysis of the kinetics of incorporation of radioactive phosphate into macromolecules showed that the periodicity of phospholipid synthesis could not have been detected by pulse-labeling techniques, and only an analysis of cells prelabeled to equilibrium allowed detection of the periodicity. Equilibrium-labeled cells also allowed determination of the absolute amount of phosphorus-containing macromolecules in newborn swarmer cells. These cells contain about as much DNA as one Escherichia coli chromosome and about four times as much RNA as DNA. The amount of phosphorus in phospholipids is about one-seventh of that in DNA, or about 3% of the total macromolecular phosphorus.
Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12
Authors: von Meyenburg K , Boye E , Skarstad K , Koppes L , Kogoma T
Published: November 2016

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.