bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of competition-defective mutants of Rhizobium fredii
Authors: McLoughlin T J , Merlo A O , Satola S W , Johansen E
Published: November 2016

We coupled Tn5 mutagenesis with a competition assay to isolate mutants of Rhizobium fredii USDA 257 that are defective in competition for nodulation of soybeans. Two mutants with single Tn5 inserts in the chromosome showed reduced competitiveness in vermiculite but were identical to the wild-type strain in symbiotic properties when inoculated alone. Recombination of Tn5 and flanking genomic regions cloned from the mutants into the parent strain showed that Tn5 was responsible for the mutant phenotype.
Nonrandom minichromosome replication in Escherichia coli K-12
Authors: Koppes L J , von Meyenburg K
Published: November 2016

The intervals between rounds of chromosome and minichromosome replication were measured by density shift experiments and found to be similar. Thus the minichromosome, a lambda asnA oriC bacteriophage, mostly replicates once each division cycle rather than randomly, despite its high copy number. Slight differences between the chromosome and the oriC plasmid are explained.
Characterization and mapping of the Bacillus subtilis prtR gene
Authors: Yang M , Shimotsu H , Ferrari E , Henner D J
Published: November 2016

A gene from Bacillus natto encoding a 60-amino-acid peptide has been previously described that, when cloned on a high-copy plasmid in B. subtilis, enhances production of alkaline protease, neutral protease, and levansucrase. An identical gene was isolated from B. subtilis and caused a similar phenotype when placed on a high-copy plasmid. Genetic mapping localized this gene near metB, distant from other pleiotropic genes causing similar effects. Deletion of this gene from the B. subtilis chromosome had no obvious phenotypic effect.
Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides
Authors: Djordjevic S P , Chen H , Batley M , Redmond J W , Rolfe B G
Published: November 2016

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.
Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli
Authors: Driehuis F , Wouters J T
Published: November 2016

The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography. At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1). The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly. Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material. In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall.
Instability of Tn5 inserts in cyanobacterial cloning vectors
Authors: Gendel S M
Published: November 2016

Transposon Tn5 was used to produce random insertions in two hybrid cloning vectors for the unicellular cyanobacterium Anacystis nidulans. The transposon-containing plasmids were used to localize essential replication functions and to characterize the stability of large inserts in these vectors. The effect of the insertions on plasmid function was tested by transformation into a derivative of A. nidulans that had been cured of the endogenous plasmid used to construct the vectors. A region of approximately 4 kilobases was essential for successful plasmid transformation and replication. This region has also been shown to be involved in plasmid replication by deletion analysis. High rates of excision of Tn5 inserts within this region and restoration of normal replication function were observed when transformants were selected by using a resistance marker outside the replication region in the absence of selection for the transposon-coded kanamycin resistance. Transposon inserts outside this region were not deleted.
Tn4556, a 6.8-kilobase-pair transposable element of Streptomyces fradiae
Authors: Chung S T
Published: November 2016

A 6.8-kilobase-pair (kbp) transposable element (Tn4556) was found in a neomycin-producing strain of Streptomyces fradiae. This element was first observed in two 30.3-kbp plasmids (pUC1123 and pUC1124) which arose when a thiostrepton resistance gene (1 kbp) was ligated with the BclI-2 fragment (22.5 kbp) that contains the origin of replication of phage SF1. The Tn4556 segment was deleted when these plasmids were transduced into another S. fradiae host with phage SF1. These deletion plasmids (pUC1210 and pUC1211) had copy numbers of less than 1 per chromosome and were unstable. In contrast, pUC1123 and pUC1124, with copy numbers of 12 to 15 per chromosome, respectively, were relatively stable. When pUC1210 and pUC1211 were reintroduced into S. fradiae by protoplast transformation, the Tn4556 element transposed again to the plasmids at numerous new locations in either of two orientations. A copy of Tn4556 was found in the S. fradiae chromosome by hybridization studies. It appears that Tn4556 originated from the chromosome, transposed into unstable pUC1210 and pUC1211, and made stable plasmids. A temperature-sensitive hybrid plasmid carrying a viomycin resistance derivative of Tn4556 (pMT660::Tn4556::vph) was constructed. When Streptomyces lividans UC8390 containing the hybrid plasmid was grown at 39 degrees C, Tn4556::vph (Tn4560) transposed to random positions in the host chromosome.
Highly repressible expression system for cloning genes that specify potentially toxic proteins
Authors: O'Connor C D , Timmis K N
Published: November 2016

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational.
Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus
Authors: Doyle C M , Arp D J
Published: November 2016

Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.
Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp
Authors: Xing R Y , Whitman W B
Published: November 2016

The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the Ki for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive, partially resistant, and resistant methanococci (Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae, respectively), the Ki for SM was 0.0012, 0.34, and greater than 1.0 mM, respectively. Inhibition was uncompetitive with respect to pyruvate. Based on these observations, ALS appeared to be the major if not the sole site of action of SM in the methanococci. The sensitivity of the ALS from these three methanococci to feedback inhibition by branched-chain amino acids was also quite different. Although all three were sensitive to feedback inhibition by valine, the Ki varied 20-fold, from 0.01 to 0.22 mM. Moreover, only the ALS from M. maripaludis was sensitive to inhibition by leucine, and the Ki was 1.8 mM. The Ki for isoleucine for the ALS from both M. maripaludis and M. voltae was about 0.1 mM. The ALS from M. aeolicus was not inhibited by isoleucine. In other respects, the ALSs from the methanococci were very similar. After dialysis, thiamine pyrophosphate but not FAD and Mg2+ was required for maximal activity, and they were all rapidly inactivated by oxygen. Although the methanococcal ALSs exhibited diverse properties, the range of catalytic and regulatory properties closely resembled those of the eubacterial enzymes.
Purification and characterization of a pectin lyase produced by Pseudomonas fluorescens W51
Authors: Schlemmer A F , Ware C F , Keen N T
Published: November 2016

A pectin lyase (PNL; EC 4.2.2.10) was isolated from culture filtrates of Pseudomonas fluorescens W51 and purified to apparent homogeneity. The enzyme catalyzed a random eliminative cleavage of pectin but not sodium polypectate, and it macerated plant tissue. The Mr of the PNL on sodium dodecyl sulfate-polyacrylamide gels was 32,000 +/- 1,000, and the isoelectric point was 9.4 as determined by isoelectric focusing. The enzyme was constitutively produced, since the highest yields were obtained when glycerol was used as a sole carbon source, and addition of pectin to the medium did not increase its production. Antibodies against purified PNL reacted in Western blots (immunoblots) with a pectate lyase (PLb) produced by Erwinia chrysanthemi EC16. The PNL appeared to be the only factor secreted into the culture medium by P. fluorescens W51 which macerated plant tissue and is probably involved in the soft rot disease caused by the bacterium.
Nucleotide sequence analysis of Tn4551: use of ermFS operon fusions to detect promoter activity in Bacteroides fragilis
Authors: Smith C J
Published: November 2016

The Bacteroides pBI136 clindamycin resistance (Ccr) determinant from the composite transposon Tn4551 was cloned onto the shuttle plasmid pFD160, and the regions necessary for expression in Bacteroides fragilis were determined. These results suggested that transcriptional regulatory signals required for Ccr were located in the Tn4551 direct repeat sequence (DRS) adjacent to the resistance determinant. Analysis of the nucleotide sequence of this region revealed that the Ccr structural gene, 798 base pairs (bp), was located 17 bp from the terminus of the DRS and that this gene (ermFS) differed from ermF (pBF4) by one amino acid. The DRS element was found to be 1,155 bp and appeared to contain the ermFS transcription start signals. The DRS structure was typical of insertion sequence elements isolated from other bacterial species, and its termini were characterized by 25-bp regions of imperfect dyad symmetry. The DRS was dominated by a 978-bp open reading frame, which terminated in the left inverted repeat 27 bp from the ermFS start codon, and weak amino acid sequence homology was observed with the putative transposase of IS3. Promoter activity of the DRS in B. fragilis was demonstrated by in vitro construction of operon fusions with a promoterless ermFS gene followed by transformation of the recombinant plasmids with selection for resistance to clindamycin. The location of one DRS promoter was identified by using the ermFS fusions and then verified by in vitro mutagenesis of the site with single-stranded linkers. Northern blot (RNA blot) analysis of total RNA from B. fragilis strains containing pBI136 or ermFS recombinant plasmids confirmed the location of this promoter and indicated that it was used in vivo by Tn4551. A second DRS promoter, which activated ermFS transcription by readthrough of the large DRS open reading frame, was also identified by the Northern blot analysis. The bicistronic ermFS message was not observed in strains containing a complete copy of Tn4551, and the possibility of transcriptional regulation is discussed.
Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa
Authors: Ostroff R M , Vasil M L
Published: November 2016

The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization. The mutant strain, designated PLC S, is nonhemolytic and lacks a 78-kilodalton protein corresponding to the size of the wild-type PLC. However, there is an additional phospholipase activity present in PLC S capable of hydrolyzing p-nitrophenylphosphorylcholine, a synthetic PLC substrate, and phosphatidylcholine. This enzymatic activity is not a result of a truncated product produced from the mutated plcS gene. The phospholipase activity of PLC S was identified as a nonhemolytic PLC.
Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica
Authors: Davidow L S , O'Donnell M M , Kaczmarek F S , Pereira D A , DeZeeuw J R , Franke A E
Published: November 2016

The XPR2 gene encoding an alkaline extracellular protease (AEP) from Yarrowia lipolytica was cloned, and its complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AEP consists of 297 amino acids with a relative molecular weight of 30,559. The gene codes for a putative 22-amino-acid prepeptide (signal sequence) followed by an additional 135-amino-acid propeptide containing a possible N-linked glycosylation site and two Lys-Arg peptidase-processing sites. The final Lys-Arg site occurs at the junction with the mature, extracellular form. The mature protease contains two potential glycosylation sites. AEP is a member of the subtilisin family of serine proteases, with 42.6% homology to the fungal proteinase K. The functional promoter is more than 700 base pairs long, allowing for the observed complex regulation of this gene. The 5' and 3' flanking regions of the XPR2 gene have structural features in common with other yeast genes.
DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli
Authors: Gaylo P J , Turjman N , Bastia D
Published: November 2016

The minimal origin of replication of the broad-host-range plasmid RK2 has two potential recognition sequences for the DnaA protein of Escherichia coli. DNA transfer by transformation into a dnaA-null mutant of E. coli showed that DnaA protein is needed for replication or maintenance of mini-RK2. We isolated and purified DnaA protein as a chimeric protein, covalently attached to a piece of collagen and beta-galactosidase. The hybrid protein specifically bound to restriction fragments from the oriV region of RK2, which contained the two dnaA boxes. Deletion of the second dnaA box inactivated the origin and abolished the binding of the hybrid protein to the DNA fragment that had suffered the deletion. When the second dnaA box was replaced with an EcoRI linker of identical length, origin activity was restored. Binding experiments showed that the linker provided a weak dnaA box. An alternative explanation was that the linker restored proper spacing between sequences on either side of the deleted box, thus restoring origin activity.
Role of micF in the tolC-mediated regulation of OmpF, a major outer membrane protein of Escherichia coli K-12
Authors: Misra R , Reeves P R
Published: November 2016

Mutation in the tolC locus greatly reduces normal synthesis of OmpF, a major porin protein of Escherichia coli K-12. Experiments that use ompF-ompC chimeric genes demonstrate that a tolC mutation exerts its effect at either the promoter or the amino-terminal end of the ompF gene. Direct analysis of ompF mRNA from tolC+ and tolC strains showed that the amount of ompF transcript in the latter was greatly reduced. We have also observed that, in addition to reducing the amount of OmpF, a tolC mutation increases the level of OmpC protein to a much greater extent than occurs in an OmpF mutant and also increases micF RNA synthesis as shown by increased beta-galactosidase synthesis in a micF-lacZ fusion strain. Based on these observations, we suggest that an increased expression of the micF gene in a tolC mutant results in the reduced expression of ompF and that a major effect of the tolC mutation may be to push the porin-regulating system to favor ompC and micF to a greater extent than under high-osmolarity conditions.
Purification and characterization of the F1 ATPase from Bacillus subtilis and its uncoupler-resistant mutant derivatives
Authors: Hicks D B , Krulwich T A
Published: November 2016

The F1 ATPase of Bacillus subtilis BD99 was extracted from everted membrane vesicles by low-ionic-strength treatment and purified by DEAE-cellulose chromatography, hydrophobic interaction chromatography, and anion-exchange high-performance liquid chromatography. The subunit structure of the enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of urea. In the absence of urea, the alpha and beta subunits comigrated and the ATPase was resolved into four bands. The mobility of the beta subunit, identified by immunoblotting with anti-beta from Escherichia coli F1, was altered dramatically by the presence of urea, causing it to migrate more slowly than the alpha subunit. The catalytic activity of the ATPase was strongly metal dependent; in the absence of effectors, the Ca2+-ATPase activity was 15- to 20-fold higher than the Mg2+ -ATPase activity. On the other hand, sulfite anion, methanol, and optimally, octylglucoside stimulated the Mg2+ -ATPase activity up to twice the level of Ca2+ -ATPase activity (specific activity, about 80 mumol of Pi per min per mg of protein). The F1 ATPase was also isolated from mutants of B. subtilis that had been isolated and characterized in this laboratory by their ability to grow in the presence of protonophores. The specific activities of the ATPase preparations from the mutant and the wild type were very similar for both Mg2+- and Ca2+ -dependent activities. Kinetic parameters (Vmax and Km for Mg-ATP) for octylglucoside-stimulated Mg2+ -ATPase activity were similar in both preparations. Structural analysis by polyacrylamide gel electrophoresis and isoelectric focusing indicated that the five F1 subunits from ATPase preparations from the mutant and wild-type strains had identical apparent molecular weights and that no charge differences were detectable in the alpha and beta subunits in the two preparations. Thus, the increased ATPase activity that had been observed in the uncoupler-resistant mutants is probably not due to a mutation in the F1 moiety of the ATPase complex.
Sequence, expression, and localization of the immunity protein for colicin M
Authors: Olschläger T , Braun V
Published: November 2016

Escherichia coli strains carrying the cmi locus on plasmids are immune against colicin M, which primarily inhibits murein biosynthesis, followed by lysis of cells. The nucleotide sequence of the cmi region was determined. It contains an open reading frame for a polypeptide with a molecular weight of 19,227. However, the major protein band observed on polyacrylamide gels after transcription and translation in an in vitro system or in minicells had an apparent molecular weight between 15,000 and 16,000. The nucleotide sequence contained internal ATG codons, two of which could serve for the synthesis of polypeptides with molecular weights of 15,349 and 15,996, respectively. A subclone with a DNA fragment that encoded these two shorter polypeptides exhibited full immunity. The colicin M immunity protein was found in the cytoplasmic membrane. The colicin M activity and immunity genes were transcribed in opposite directions. Both properties are typical of the channel-forming colicins and are in contrast to the colicins with endonuclease activities. However, colicin M does not form channels and exhibits no structural similarity to channel-forming colicins.
Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content
Authors: Ohama T , Yamao F , Muto A , Osawa S
Published: November 2016

The DNA sequence of the Micrococcus luteus str operon, which includes genes for ribosomal proteins S12 (str or rpsL) and S7 (rpsG) and elongation factors (EF) G (fus) and Tu (tuf), has been determined and compared with the corresponding sequence of Escherichia coli to estimate the effect of high genomic G + C content (74%) of M. luteus on the codon usage pattern. The gene organization in this operon and the deduced amino acid sequence of each corresponding protein are well conserved between the two species. The mean G + C content of the M. luteus str operon is 67%, which is much higher than that of E. coli (51%). The codon usage pattern of M. luteus is very different from that of E. coli and extremely biased to the use of G and C in silent positions. About 95% (1,309 of 1,382) of codons have G or C at the third position. Codon GUG is used for initiation of S12, EF-G, and EF-Tu, and AUG is used only in S7, whereas GUG initiates only one of the EF-Tu's in E. coli. UGA is the predominant termination codon in M. luteus, in contrast to UAA in E. coli.
Membrane topography of anaerobic carbon monoxide oxidation in Rhodocyclus gelatinosus
Authors: Champine J E , Uffen R L
Published: November 2016

Rhodocyclus gelatinosus 1 grows anaerobically in the dark at the expense of carbon monoxide. Topographical studies with methyl viologen as the membrane probe indicated that CO oxidation and H2 production sites were on the cytoplasmic side of the cell membrane. Membrane-associated hydrogen gas production appeared to be a unidirectional reaction. In the dark, strain 1 whole cells oxidized CO and incorporated about 306 pmol of 32Pi into ATP per min per mg of protein. With CO as the sole energy-yielding substrate, cells grew with a low growth yield coefficient of 3.7 g (dry weight) of cells per mg of CO oxidized.
recA gene of Escherichia coli complements defects in DNA repair and mutagenesis in Streptomyces fradiae JS6 (mcr-6)
Authors: Matsushima P , Baltz R H
Published: November 2016

Streptomyces fradiae JS6 (mcr-6) is a mutant which is defective in repair of DNA damage induced by a variety of chemical mutagens and UV light. JS6 is also defective in error-prone (mutagenic) DNA repair (J. Stonesifer and R. H. Baltz, Proc. Natl. Acad. Sci. USA 82:1180-1183, 1985). The recA gene of Escherichia coli, cloned in a bifunctional vector that replicates in E. coli and Streptomyces spp., complemented the mutation in S. fradiae JS6, indicating that E. coli and S. fradiae express similar SOS responses and that the mcr+ gene product of S. fradiae is functionally analogous to the protein encoded by the recA gene of E. coli.
Cloning and expression in Escherichia coli of chromosomal mercury resistance genes from a Bacillus sp
Authors: Wang Y , Mahler I , Levinson H S , Halvorson H O
Published: November 2016

A 7.9-kilobase (kb) chromosomal fragment was cloned from a mercury-resistant Bacillus sp. In Escherichia coli, in the presence of a second plasmid carrying functional transport genes, resistance to HgCl2 and to phenylmercury acetate (PMA) was expressed. Shortening the cloned fragment to 3.8 kb abolished resistance to PMA but not to HgCl2. In Bacillus subtilis, the 3.8-kb fragment produced mercuric reductase constitutively but did not produce resistance to HgCl2 or to PMA.
Structure and organization of the hisA gene of the thermophilic archaebacterium Methanococcus thermolithotrophicus
Authors: Weil C F , Beckler G S , Reeve J N
Published: November 2016

A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli. Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M. thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M. voltae and M. vannielii, respectively. The ORF (ORF 206) immediately 5' to the hisA gene of M. thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.
Penicillin-binding protein 7 and its relationship to lysis of nongrowing Escherichia coli
Authors: Tuomanen E , Schwartz J
Published: November 2016

The characteristics and possible physiological role(s) of a 30-kilodalton low-molecular-size penicillin-binding protein (PBP), PBP 7, in Escherichia coli were investigated. The amount of penicillin required to half saturate PBP 7 was approximately 5 micrograms/ml, and this PBP bound 15% of the total penicillin bound to all PBPs with a deacylation rate of greater than 120 min. This PBP was distinguishable from E. coli PBPs 1 to 6 by the pattern of [3H]penicillin-labeled peptides generated by partial proteolysis. A unique feature of PBP 7 was its capacity to bind numerous members of penem class antibiotics at relatively low concentrations. A striking correlation existed in drugs between capability of lysing amino acid-starved (nongrowing) E. coli and binding to PBP 7. The findings suggest that PBP 7 is a new, physiologically significant target for beta-lactam antibiotics.
Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans
Authors: Sugio T , Mizunashi W , Inagaki K , Tano T
Published: November 2016

A sulfur:ferric ion oxidoreductase that utilizes ferric ion (Fe3+) as an electron acceptor of elemental sulfur was purified from iron-grown Thiobacillus ferrooxidans to an electrophoretically homogeneous state. Under anaerobic conditions in the presence of Fe3+, the enzyme reduced 4 mol of Fe3+ with 1 mol of elemental sulfur to give 4 mol of Fe2+ and 1 mol of sulfite, indicating that it corresponds to a ferric ion-reducing system (T. Sugio, C. Domatsu, O. Munakata, T. Tano, and K. Imai, Appl. Environ. Microbiol. 49:1401-1406, 1985). Under aerobic conditions, sulfite, but not Fe2+, was produced during the oxidation of elemental sulfur by this enzyme because the Fe2+ produced was rapidly reoxidized chemically by molecular oxygen. The possibility that Fe3+ serves as an electron acceptor under aerobic conditions was ascertained by adding o-phenanthroline, which chelates Fe2+, to the reaction mixture. Sulfur:ferric ion oxidoreductase had an apparent molecular weight of 46,000, and it is composed of two identical subunits (Mr = 23,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sulfur oxidation by this enzyme was absolutely dependent on the presence of reduced glutathione. The enzyme had an isoelectric point and a pH optimum at pH 4.6 and 6.5, respectively. Almost all the activity of sulfur:ferric ion oxidoreductase was observed in the osmotic shock fluid of the cells, suggesting that it was localized in the periplasmic space of the cells.
Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development
Authors: Carlson R W , Kalembasa S , Turowski D , Pachori P , Noel K D
Published: November 2016

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.
Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli
Authors: Wachi M , Doi M , Tamaki S , Park W , Nakajima-Iijima S , Matsuhashi M
Published: November 2016

A chromosomal region of Escherichia coli contiguous to the fabE gene at 71 min on the chromosomal map contains multiple genes that are responsible for determination of the rod shape and sensitivity to the amidinopenicillin mecillinam. The so-called mre region was cloned and analyzed by complementation of two closely related but distinct E. coli mutants characterized, respectively, by the mutations mre-129 and mre-678, that showed a rounded to irregular cell shape and altered sensitivities to mecillinam; the mre-129 mutant was supersensitive to mecillinam at 30 degrees C, but the mre-678 mutant was resistant. The mre-678 mutation also caused simultaneous overproduction of penicillin-binding proteins 1Bs and 3. A chromosomal region of the wild-type DNA containing the total mre region and the fabE gene was first cloned on a lambda phage; a 7-kilobase (kb) fragment containing the whole mre region, but not the fabE gene, was then recloned on a mini F plasmid, pLG339; and finally, a 2.8-kb fragment complementing only mre-129 was also cloned on this low-copy-number plasmid. The whole 7-kb fragment was required for complementing the mre-678 mutant phenotypes. Fragments containing fabE but not the mre-129 region could be cloned on a high-copy-number plasmid. Southern blot hybridization indicated that the mre-678 mutant had a large deletion of 5.25 kb in its DNA, covering at least part of the mre-129 gene.
Purification and properties of alpha-pinene oxide lyase from Nocardia sp. strain P18.3
Authors: Griffiths E T , Harries P C , Jeffcoat R , Trudgill P W
Published: November 2016

alpha-Pinene oxide is an intermediate in the degradation of alpha-pinene by Nocardia sp. strain P18.3 and some Pseudomonas strains. The epoxide is cleaved by a lyase which catalyzes a concerted reaction in which both rings of the bicyclic structure are cleaved with the formation of cis-2-methyl-5-isopropylhexa-2,5-dienal. The enzyme has been purified to homogeneity from Nocardia sp. strain P18.3. It was induced by growth with alpha-pinene and constituted 6 to 7% of the soluble protein of cell extracts. The apparent molecular weight of the native enzyme was 50,000 by ultracentrifugal analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave two dissimilar subunits with apparent molecular weights of 17,000 and 22,000. The enzyme was devoid of prosthetic groups, had no cofactor requirement, and had a broad pH activity range, a Km for alpha-pinene oxide of 9 microM, and a turnover number of 15,000. Inhibitors included sulfhydryl reactive compounds, terpene epoxides, and pinane derivatives with substituent groups at carbon 3. A mechanism for the concerted reaction has been proposed in which decyclization is initiated by donation of a proton from the catalytic center to the oxygen of the epoxide with consequent destabilization. In vitro the enzyme was inactivated during catalysis, and a reactive cationic intermediate may be responsible for this phenomenon. The enzyme should be classified as a lyase EC 4.99.-.-.
Parasexual genetics of Torulopsis glabrata
Authors: Whelan W L , Kwon-Chung K J
Published: November 2016

Prototrophic hybrids were generated in the asexual yeast Torulopsis glabrata by the fusion of spheroplasts derived from parent strains which bore complementing auxotrophic markers. The DNA content (per cell) of two hybrids was essentially that predicted by summing the corresponding parental values. UV irradiation of these two hybrids resulted in the formation of sectored colonies with genetic properties consistent with their origin by either mitotic recombination or chromosomal nondisjunction.
Effects of intracellular trehalose content on Streptomyces griseus spores
Authors: McBride M J , Ensign J C
Published: November 2016

The disaccharide trehalose is accumulated as a storage product by spores of Streptomyces griseus. Growth on media containing excess glucose yielded spores containing up to 25% of their dry weight as trehalose. Spores containing as little as 1% of their dry weight as trehalose were obtained during growth on media containing a limiting amount of glucose. Spores containing low levels of trehalose accumulated this sugar when incubated with glucose. The increase in trehalose content coincided with increases in spore refractility, heat resistance, desiccation resistance, and the time required for spore germination in complex media. Trehalose is accumulated by a wide variety of actinomycetes and related bacteria and may be partially responsible for their resistance properties.
Genetics of type IIa heat-labile enterotoxin of Escherichia coli: operon fusions, nucleotide sequence, and hybridization studies
Authors: Pickett C L , Weinstein D L , Holmes R K
Published: November 2016

Operon fusions for the Escherichia coli heat-labile enterotoxin type IIa (LT-IIa) operon were isolated and characterized. The LT-IIa genes are organized in a transcriptional unit similar to those of cholera toxin (CT) and the closely related E. coli heat-labile toxin type I (LT-I, with subtypes LTh-I and LTp-I). The nucleotide sequence of the LT-IIa genes was determined and compared with the sequences of LTh-I and CT. The A subunit gene of LT-IIa was found to be 57% homologous with the A subunit gene of LTh-I and 55% homologous with the A gene of CT. Most of the homology derived from the region of the A gene which encodes the A1 fragment. The B gene of LT-IIa was not homologous with the B gene of LTh-I or CT. DNA probes containing various portions of the LT-IIa genes and adjacent sequences were used for hybridization studies with restriction endonuclease fragments of DNA from a collection of LT-II-producing strains. These studies showed that a probe containing much of the A subunit gene hybridized well to DNA from the various strains, but a probe for the B subunit gene did not.
Genetic and physical location of the Escherichia coli rap locus, which is essential for growth of bacteriophage lambda
Authors: Guarneros G , Machado G , Guzmán P , Garay E
Published: November 2016

The Escherichia coli rap mutant does not support the growth of bacteriophage lambda (D. Henderson and J. Weil, Virology 71:546-559, 1976). We located the rap site at 26 min in the E. coli genetic map and determined the gene order fadR-rap-supF-trp from our transduction experiments. Plasmid pHO1 harbors a 5.6-kilobase-pair segment of the E. coli chromosome which contains the pth gene (B. Hove-Jensen, Mol. Gen. Genet. 201:269-276, 1985). This plasmid complemented rap bacteria, suggesting that it carries the dominant allele rap+. Subcloning experiments reduced the rap-complementing segment to 1.5 kilobase pairs. This segment still contained pth; thus, both loci are tightly linked. The lit mutations that inhibit phage T4 growth in E. coli are located nearby at 25 min (W. Cooley, K. Sirotkin, R. Green, and L. Snyder, J. Bacteriol. 140:83-91, 1979). We showed that rap and lit mutations are phenotypically and genetically different.
Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies
Authors: Peters J , Peters M , Lottspeich F , Schäfer W , Baumeister W
Published: November 2016

The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.
Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies
Authors: Reddy A , Battisti L , Thorne C B
Published: November 2016

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.
Identification of Treponema pallidum penicillin-binding proteins
Authors: Cunningham T M , Miller J N , Lovett M A
Published: November 2016

Penicillin-binding proteins of 180, 89, 80, 68, 61, 41, and 38 kilodaltons were identified in Treponema pallidum (Nichols) by their covalent binding of [35S]benzylpenicillin. Penicillin-binding proteins are localized in the plasma membranes of many bacterial species and may serve as useful markers for determining plasma membrane intactness in T. pallidum fractionation studies.
Effects of segregation and selection on instability of plasmid pACYC184 in Escherichia coli B
Authors: Lenski R E , Bouma J E
Published: November 2016

We use a mathematical model to analyze the dynamics of loss of nonconjugative pACYC184 from populations of Escherichia coli B in glucose-limited continuous culture. This model incorporates both plasmid segregation and selection against plasmid carriage. It is concluded that there is intense selection against plasmid carriage (s = 0.3 per culture generation), which amplifies the frequency of segregants arising de novo.
Gene transfer from Escherichia coli to Campylobacter species: development of shuttle vectors for genetic analysis of Campylobacter jejuni
Authors: Labigne-Roussel A , Harel J , Tompkins L
Published: November 2016

A shuttle cloning vector (pIL550) has been constructed which can be mobilized from Escherichia coli to Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by complementation with the transfer functions of an IncP plasmid in trans, with a frequency of 10(-4) transconjugants per donor. We also present evidence for a DNA modification system in C. jejuni.
Pathway of acetate assimilation in autotrophic and heterotrophic methanococci
Authors: Shieh J S , Whitman W B
Published: November 2016

The autotroph Methanococcus maripaludis contained high levels of acetate-coenzyme A ligase, pyruvate synthase, pyruvate, water dikinase, pyruvate carboxylase, and the enzymes of the incomplete reductive tricarboxylic acid cycle. Phosphoenolpyruvate carboxykinase, citrate synthase, and isocitrate dehydrogenase were not detected. In contrast, the heterotroph Methanococcus sp. strain A3 contained acetate kinase, and acetate coenzyme A ligase was virtually absent.
Two single-base-pair substitutions causing desensitization to tryptophan feedback inhibition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum
Authors: Matsui K , Miwa K , Sano K
Published: November 2016

A 5-fluorotryptophan-resistant mutant, termed 1041, was isolated from Brevibacterium lactofermentum AJ12036. The anthranilate synthase of 1041 was insensitive to feedback inhibition by tryptophan, and the specific activities of the anthranilate synthase and anthranilate phosphoribosyltransferase of 1041 were 29- and 23-fold higher than those in parental strain AJ12036, respectively. A single-base change (adenine to cytosine) that resulted in a Ser-to-Arg substitution was found in the trpE structural gene of 1041. This substitution was identified as the cause of the desensitization to feedback inhibition by tryptophan of anthranilate synthase in 1041. Another substitution (guanine to adenine) was found at a position in which a mutation would destabilize the rho-independent terminator structure within the putative attenuator. The enhanced synthesis of tryptophan enzymes in 1041 could be caused by this substitution in the attenuator.
Efficient utilization and operation of the gluconate-inducible system of the promoter of the Bacillus subtilis gnt operon in Escherichia coli
Authors: Miwa Y , Fujita Y
Published: November 2016

A DNA fragment containing the promoter of the Bacillus subtilis gluconate (gnt) operon and its first gene (gntR) was cloned into Escherichia coli. E. coli recognized this promoter efficiently and precisely. Moreover, the gluconate-inducible system of this operon operated even in E. coli.
Chemotaxis to plant phenolic inducers of virulence genes is constitutively expressed in the absence of the Ti plasmid in Agrobacterium tumefaciens
Authors: Parke D , Ornston L N , Nester E W
Published: November 2016

The virulence (vir) genes are required in the early stages of plant tumor formation and are located together on the tumor-inducing (Ti) plasmid in Agrobacterium tumefaciens. Five of the vir genes are expressed inducibly in response to the following monocyclic phenolic compounds: acetosyringone, catechol, gallate, beta-resorcylate, protocatechuate, p-hydroxybenzoate, and vanillin. Of these compounds, only the latter six, excluding vanillin [corrected] served as chemoattractants and only the latter three served as growth substrates for A. tumefaciens A348. Strain A136, isogenic except for lack of the Ti plasmid, demonstrated chemotactic behavior and nutritional capabilities similar to those of strain A348. The chemotactic response to the vir gene inducers was expressed constitutively.
Cloning and promoter identification of the iron-regulated cir gene of Escherichia coli
Authors: Griggs D W , Tharp B B , Konisky J
Published: November 2016

The cir gene, which encodes the colicin I receptor protein and is regulated by both cellular iron content and growth temperature, was cloned into a multicopy-number plasmid. Physical mapping and complementation analysis established the position of cir between mgl and nfo on the Escherichia coli chromosome. A gene encoding a 32,000-dalton polypeptide was located downstream of and adjacent to cir, but did not appear to be part of the same transcriptional unit. A 525-base-pair fragment from the 5' end of the 1.8-kilobase-pair receptor-coding region directed iron-regulated transcription and translation of a hybrid cir-lacZ gene. Two overlapping promoters were identified by determination of the transcriptional start sites and by sequence analysis. A small open reading frame (120 nucleotides) of unknown significance preceded the receptor-coding sequence. Examination of the amino acid sequence of the receptor purified from the outer membrane revealed that the gene product was processed by removal of a signal peptide and that the mature form had an amino acid sequence near its amino terminus which closely resembled that of several other TonB-dependent proteins.