bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition
Authors: Eoyang L , Silverman P M
Published: November 2016

Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10-fold reduction in enzyme specific activity.
Selection by genetic transformation of a Saccharomyces cerevisiae mutant defective for the nuclear uracil-DNA-glycosylase
Authors: Burgers P M , Klein M B
Published: November 2016

A coliphage M13 chimer containing the Saccharomyces cerevisiae TRP1 gene and ARS1 replication origin (mPY2) was grown on an ung- dut- strain of Escherichia coli. The resulting single-stranded phage DNA had 13% of thymine residues substituted by uracil. This DNA failed to transform a delta trp1 yeast strain to prototrophy. However, when a mutagenized yeast stock was transformed with uracil-containing single-stranded mPY2 DNA, unstable transformants were obtained. After plasmid segregation, about half of these were retransformed at a high frequency by uracil-containing single-stranded mPY2 DNA. In vitro, these mutants were defective for uracil-DNA-glycosylase activity. They were designated ung1. Strains containing the ung1 mutation have an increased sensitivity to sodium bisulfite and sodium nitrite but a wild-type sensitivity to methyl methanesulfonate, UV light, and drugs that cause depletion of the thymidylate pool. They have a moderate mutator phenotype for nuclear but not for mitochondrial genes. A low mitochondrial uracil-DNA-glycosylase activity was demonstrated in the mutant strains.
Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake
Authors: Pierce J R , Earhart C F
Published: November 2016

Escherichia coli genes specifically required for transport of iron by the siderophore enterobactin are designated fep. The studies reported here were initiated to identify and localize the fepB product. The plasmid pCP111, which consisted of an 11-kilobase E. coli DNA fragment containing fepB ligated to pACYC184, was constructed. The fepB gene was subcloned; in the process, complementation tests and Tn5 mutagenesis results provided evidence for the existence of a new fep gene, fepC. The order of the transport genes in the ent gene cluster is as follows: fepA fes entF fepC fepB entE. Minicell, maxicell, and in vitro DNA-directed protein synthesizing systems were used to identify the fepB and fepC products. The fepC polypeptide was 30,500 daltons in standard sodium dodecyl sulfate-polyacrylamide gels. The fepB gene was responsible for the appearance of three or four bands (their apparent molecular weights ranged from 31,500 to 36,500) in sodium dodecyl sulfate-polyacrylamide gels, depending on the gel system employed. The largest of these was tentatively designated proFepB, since it apparently had a leader sequence. Localization experiments showed that FepC was a membrane constituent and that mature FepB was present in the periplasm. An additional polypeptide (X) was also encoded by the bacterial DNA of pCP111, but its relationship to iron transport is unknown. The results indicated that ferrienterobactin uptake is mediated by a periplasmic transport system and that genes coding for outer membrane (fepA), periplasmic (fepB), and cytoplasmic membrane (fepC) components have now been identified.
Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron
Authors: Guthrie E P , Salyers A A
Published: November 2016

Bacteroides thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. Both enzymes have very similar biochemical properties. To determine whether both enzymes are required for growth on chondroitin sulfate, we constructed a Bacteroides suicide vector, pE3-1, and used it to create an insertional mutation that interrupts the chondroitin lyase II gene of Bacteroides thetaiotaomicron. pE3-1 contains a 4.4-kilobase cryptic B. eggerthii plasmid (pB8-51), the Escherichia coli cloning vector pBR328, and the EcoRI D fragment from the conjugative B. fragilis plasmid pBF4. A 0.8-kilobase fragment from the center of the B. thetaiotaomicron chondroitin lyase II gene was inserted in pE3-1 to create pEG817. Although, pEG817 is stably maintained in E. coli and can be mobilized into B. thetaiotaomicron by the IncP plasmid R751, pEG817 is not maintained as a plasmid in Bacteroides spp. When pEG817 was mobilized into B. thetaiotaomicron, with selection for a drug marker on pEG817, transconjugants were obtained which had pEG817 inserted into the chondroitin lyase II gene. Western blot analysis was used to confirm that intact chondroitin lyase II is not produced in the mutant. The mutant was able to utilize chondroitin sulfate as a sole source of carbon, although no active chondroitin lyase II was produced. Thus chondroitin lyase I alone appears to be sufficient for growth on chondroitin sulfate. The mutant also had some minor changes in its outer membrane protein profile. However, there was no evidence that any of the major chondroitin sulfate-associated polypeptides in the outer membrane were affected by the insertion in the chondroitin lyase II gene.
Interaction of FtsA and PBP3 proteins in the Escherichia coli septum
Authors: Tormo A , Ayala J A , de Pedro M A , Aldea M , Vicente M
Published: November 2016

Mutations in the ftsA gene of Escherichia coli conferred a higher resistance to lysis induced by penicillin or by a combination of cefsulodin and furazlocillin. The ftsA2 allele codes for an FtsA protein which is inactive at 42 degrees C but is able to regain its activity once it is transferred back to 30 degrees C; ftsA2 filaments formed at 42 degrees C in the presence of penicillin divided once the penicillin was removed and the temperature was lowered to 30 degrees C. Potential septation sites in the filaments of wild-type cells treated in the same way remained inactive. The binding of a radioactively labeled derivative of ampicillin to penicillin-binding protein 3 (PBP3) was significantly decreased in strain D-3, containing the mutant allele ftsA3, when the binding assay was performed at the restrictive temperature. A molecular species able to cross-react with an anti-PBP3 serum was nevertheless found to be present in the envelope of D-3 cells. These observations suggested that the FtsA protein, a protein with a structural and regulatory role in septation, and PBP3, a protein enzymatically active in the synthesis of murein for septation, interact with each other.
5'-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis
Authors: Aymerich S , Gonzy-Tréboul G , Steinmetz M
Published: November 2016

The regulation of the levansucrase gene sacB was studied in Bacillus subtilis strains. Fusions were constructed in which genes of cytoplasmic proteins such as lacZ were placed immediately downstream from sacR, the regulatory region located upstream from sacB. These fusions were introduced in mutants affected in sacB regulation. In all cases the marker gene was affected in the same way as sacB by the genetic context. This result is of particular interest for the sacU pleiotropic mutations, which affect sacB expression and other cellular functions such as the synthesis of several exocellular enzymes. We also showed that strains harboring sacU+ or sacU-hyperproducing alleles contained different amounts of sacB mRNA, which was proportional to their levansucrase secretion. We concluded that the sacU gene does not affect sacB expression at the level of secretion but acts on a target within sacR. We discuss the possibility that sacU acts on a part of sacR, a homologous copy of which was found upstream from the gene of another sacU-dependent secreted enzyme of B. subtilis, beta-glucanase.
Solubilization, purification, and characterization of succinate dehydrogenase from membranes of Mycobacterium phlei
Authors: Reddy T L , Weber M M
Published: November 2016

Succinate dehydrogenase (SDH) was solubilized from membranes of Mycobacterium phlei by Triton X-100 with a recovery of about 90%. The solubilized SDH was purified about 90-fold by Sephacryl S-300, DEAE-cellulose, hydroxylapatite, and isoelectric focusing in the presence of Triton X-100 with a 20% recovery. SDH was homogeneous, as determined by polyacrylamide gel electrophoresis in nondenaturing gels containing Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme revealed two subunits with molecular weights of 62,000 and 26,000. SDH is a flavoprotein containing 1 mol of flavin adenine dinucleotide, 7 to 8 mol of nonheme iron, and 7 to 8 mol of acid-labile sulfide per mol of protein. Using phenazine methosulfate and 2,6-dichloroindophenol as electron acceptors, the enzyme had an apparent Km of 0.12 mM succinate. SDH exhibited a sigmoidal relationship of rate to succinate concentration, indicating cooperativity. The enzyme was competitively inhibited by fumarate with a Ki of 0.15 mM. In the absence of Triton X-100, the enzyme aggregated, retained 50% of the activity, and could be resolubilized with Triton X-100 with full restoration of activity. Cardiolipin had no effect on the enzyme activity in the absence of Triton X-100, but it stimulated the activity by about 30% in the presence of 0.1% Triton X-100 in the assay mixture. Menaquinone-9(2H), isolated from M. phlei, had no effect on the enzyme activity either in the presence or absence of Triton X-100.
Evidence of homology between the pectate lyase-encoding pelB and pelC genes in Erwinia chrysanthemi
Authors: Schoedel C , Collmer A
Published: November 2016

The genes for two of several pectate lyase isozymes produced by the phytopathogenic enterobacterium Erwinia chrysanthemi 1237 were subcloned and compared by DNA-DNA hybridization, and the encoded proteins were analyzed. The borders of the genes were located on a restriction map by incremental exonuclease III deletions. DNA-DNA hybridization studies revealed a low percentage of mismatch (7 to 17%) between pelB and pelC. No homology was detected between pelC and other regions of the E. chrysanthemi 1237 chromosome, in which three other isozyme genes apparently reside. The pectate lyase isozymes were readily purified by chromatofocusing or granulated-gel bed isoelectric focusing from the periplasmic shock fluids of Escherichia coli subclones. The molecular weights of PLb and PLc were 30,000 and 33,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their isoelectric points were 7.6 and 8.1, respectively, as determined by equilibrium isoelectric focusing in ultrathin polyacrylamide gels. The Km values for PLb and PLc were 0.20 and 0.32 mg/ml, respectively, with polygalacturonate as a substrate. Thin-layer chromatography of reaction products and viscometric assays revealed little difference between the two isozymes. All our data indicate that the genes are duplicates and that the proteins are isofunctional.
Role of apparent membrane growth initiation sites during photosynthetic membrane development in synchronously dividing Rhodopseudomonas sphaeroides
Authors: Reilly P A , Niederman R A
Published: November 2016

Sites of intracytoplasmic membrane growth and temporal relations in the assembly of photosynthetic units were examined in synchronously dividing Rhodopseudomonas sphaeroides cells. After rate-zone sedimentation of cell-free extracts, apparent sites of initiation of intracytoplasmic membrane growth formed an upper pigmented band that sedimented more slowly than the intracytoplasmic membrane-derived chromatophore fraction. Throughout the cell cycle, the levels of the peripheral B800-850 light-harvesting pigment-protein complex relative to those of the core B875 complex in the upper pigmented fraction were only about half those of chromatophores. Pulse-labeling studies with L-[35S]methionine indicated that the rates of assembly of proteins in the upper pigmented fraction were much higher than those of chromatophores throughout the cell cycle; rates for the reaction center polypeptides were estimated to be approximately 3.5-fold higher than in chromatophores when the two membrane fractions were equalized on a protein basis. In pulse-chase studies, radioactivity of the reaction center and B875 polypeptides increased significantly in chromatophores and decreased in the upper pigmented band during cell division. These data suggest that the B875 reaction center cores of the photosynthetic units are inserted preferentially into sites of membrane growth initiation isolated in the upper pigmented band and that the incomplete photosynthetic units are transferred from their sites of assembly into the intracytoplasmic membrane during cell division. These results suggested further that B800-850 is added directly to the intracytoplasmic membrane throughout the cell cycle.
Bacteriophage-resistant mutants of Bacillus thuringiensis with decreased virulence in pupae of Hyalophora cecropia
Authors: Heierson A , Sidén I , Kivaisi A , Boman H G
Published: November 2016

Starting from a crystal-negative parental strain of Bacillus thuringiensis, we isolated certain bacteriophage-resistant mutants which showed decreased virulence in pupae of the cecropia moth (Hyalophora cecropia). These strains (class I mutants) were highly pleiotropic and showed resistance to seven or eight different phages, sensitivity to methicillin, and loss of flagella. They were also more sensitive to cecropia immune hemolymph in vitro. In addition, the export of at least three proteins was reduced. Revertants (class II mutants) were sensitive to phages, virulent, and resistant to penicillin derivatives. One class II mutant was a complete revertant in all properties examined. The other class II mutant was an incomplete revertant still susceptible to immune hemolymph and with repressed export of proteins. Virulence was not coupled to phage resistance as such or to lack of flagella because other mutants affected in these properties were virulent. Other factors which could be excluded as causes of virulence were production of extracellular protease and hemolysin.
Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici")
Authors: Whitehead T R , Rabinowitz J C
Published: November 2016

The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome.
Growth kinetics of individual Bacillus subtilis cells and correlation with nucleoid extension
Authors: Burdett I D , Kirkwood T B , Whalley J B
Published: November 2016

The growth rate of individual cells of Bacillus subtilis (doubling time, 120 min) has been calculated by using a modification of the Collins-Richmond principle which allows the growth rate of mononucleate, binucleate, and septate cells to be calculated separately. The standard Collins-Richmond equation represents a weighted average of the growth rate calculated from these three major classes. Both approaches strongly suggest that the rate of length extension is exponential. By preparing critical-point-dried cells, in which major features of the cell such as nucleoids and cross-walls can be seen, it has also been possible to examine whether nucleoid extension is coupled to length extension. Growth rates for nucleoid movement are parallel to those of total length extension, except possibly in the case of septate cells. Furthermore, by calculating the growth rate of various portions of the cell surface, it appears likely that the limits of the site of cylindrical envelope assembly lie between the distal tips of the nucleoid; the old poles show zero growth rate. Coupling of nucleoid extension with increase of cell length is envisaged as occurring through an exponentially increasing number of DNA-surface attachment sites occupying most of the available surface.
Methyl sterol and cyclopropane fatty acid composition of Methylococcus capsulatus grown at low oxygen tensions
Authors: Jahnke L L , Nichols P D
Published: November 2016

Methylococcus capsulatus contained extensive intracytoplasmic membranes when grown in fed-batch cultures over a wide range of oxygen tensions (0.1 to 10.6%, vol/vol) and at a constant methane level. Although the biomass decreased as oxygen levels were lowered, consistently high amounts of phospholipid and methyl sterol were synthesized. The greatest amounts of sterol and phospholipid were found in cells grown between 0.5 and 1.1% oxygen (7.2 and 203 mumol/g [dry weight], respectively). While sterol was still synthesized in significant amounts in cells grown at 0.1% oxygen, the major sterol product was the dimethyl form. Analysis by capillary gas chromatography-mass spectrophotometry showed that the phospholipid esterified fatty acids were predominantly 16:0 and 16:1 and that the hexadecenoates consisted of cis delta 9, delta 10, and delta 11 isomers. At low oxygen tensions, the presence of large amounts (25%) of cyclopropane fatty acids (cy 17:0) with the methylene groups at the delta 9, delta 10, and delta 11 positions was detected. Although the delta 9 monoenoic isomer was predominant, growth at low oxygen levels enhanced the synthesis of the delta 10 isomers of 16:1 and cy 17:0. As the oxygen level was increased, the amount of cyclopropanes decreased, such that only a trace of cy 17:0 could be detected in cells grown at 10.6% oxygen. Although M. capsulatus grew at very low oxygen tensions, this growth was accompanied by changes in the membrane lipids.
Expression of the unassembled capsid protein during infection of Shigella sonnei by bacteriophage T7 results in DNA damage that is repairable by bacteriophage T3, but not T7, DNA ligase
Authors: Beck P J , Condreay J P , Molineux I J
Published: November 2016

The abortive infection of bacteriophage T7 in Shigella sonnei D2 371-48 is characterized by a premature inhibition of phage DNA replication and nucleolytic breakdown of all phage DNA. Mutations in T7 gene 10 which are recessive to the presence of the wild-type allele can alleviate the restriction of phage growth. Phage T3 productively infects S. sonnei D2 371-48, as does a T7-T3 hybrid phage that contains, in particular, a gene 10 of T7 origin. It is the presence of T3 DNA ligase that allows phage growth on S. sonnei D2 371-48, and this enzyme can also rescue wild-type T7 from the abortive infection. T7+ is therefore functionally ligase deficient during the infection of S. sonnei D2 371-48; this deficiency is a result of the expression of the phage capsid protein, but it is independent of the assembly of the protein into a procapsid or other morphogenetic structure.
In vivo formation of hybrid aspartate transcarbamoylases from native subunits of divergent members of the family Enterobacteriaceae
Authors: Foltermann K F , Beck D A , Wild J R
Published: November 2016

The genes encoding the catalytic (pyrB) and regulatory (pyrI) polypeptides of aspartate transcarbamoylase (ATCase, EC 2.1.3.2) from several members of the family Enterobacteriaceae appear to be organized as bicistronic operons. The pyrBI gene regions from several enteric sources were cloned into selected plasmid vectors and expressed in Escherichia coli. Subsequently, the catalytic cistrons were subcloned and expressed independently from the regulatory cistrons from several of these sources. The regulatory cistron of E. coli was cloned separately and expressed from lac promoter-operator vectors. By utilizing plasmids from different incompatibility groups, it was possible to express catalytic and regulatory cistrons from different bacterial sources in the same cell. In all cases examined, the regulatory and catalytic polypeptides spontaneously assembled to form stable functional hybrid holoenzymes. This hybrid enzyme formation indicates that the r:c domains of interaction, as well as the dodecameric architecture, are conserved within the Enterobacteriaceae. The catalytic subunits of the hybrid ATCases originated from native enzymes possessing varied responses to allosteric effectors (CTP inhibition, CTP activation, or very slight responses; and ATP activation or no ATP response). However, each of the hybrid ATCases formed with regulatory subunits from E. coli demonstrated ATP activation and CTP inhibition, which suggests that the allosteric control characteristics are determined by the regulatory subunits.
Nucleotide sequence and expression of a phosphate-regulated gene encoding a secreted hemolysin of Pseudomonas aeruginosa
Authors: Pritchard A E , Vasil M L
Published: November 2016

A 3.3-kilobase-pair fragment of Pseudomonas aeruginosa DNA containing the phospholipase C (heat-labile hemolysin) gene was sequenced, and the location of the gene was determined. The gene product contains at its NH2 terminus a 38-amino acid sequence which structurally resembles the signal peptides of other secreted proteins but is unusually long and positively charged (6+). The location of the translation start codon was determined by constructing a series of plasmids in which the promoter of a transcription vector was ligated to Pseudomonas DNA containing deletions at the 5' end of the gene. The plasmids were used to transform Escherichia coli, and the resulting clones were assayed for hemolysin activity. In addition, sizes of truncated proteins produced by mutants with translation terminators introduced at specific sites were analyzed in E. coli maxicells. The gene is transcribed, starting just upstream of the hemolysin gene, as an mRNA of approximately 2,800 bases. Analysis of the nucleotide sequence, analysis of mutants in maxicells, and transcriptional studies indicate that the hemolysin is part of an operon composed of two genes. Phosphate regulation of the operon is at the transcriptional level. The location of the 5' end of the transcript was determined by S1 mapping.
Manganese reduction by a marine Bacillus species
Authors: de Vrind J P , Boogerd F C , de Vrind-de Jong E W
Published: November 2016

Mature dormant spores of marine Bacillus sp. strain SG1 catalyze the oxidation of Mn(II) to MnO2. We report that vegetative cells of the same strain reduced MnO2 under low-oxygen conditions. The rate of reduction was a function of cell concentration. The process had a pH optimum of 7.5 to 8.0 and was inhibited by HgCl2, by preheating of the cells at 80 degrees C for 5 min, by antimycin A, and by N-heptyl-hydroxy-quinoline-N-oxide. At a nonlimiting O2 concentration, little MnO2 reduction was observed. Under these conditions, the process could be induced by the addition of NaN3. Spectrophotometric analysis of the Bacillus cells indicated the presence of type b and c cytochromes. Both types can be oxidized in situ by addition of MnO2 to the cells.
Integration of photosensory signals in Halobacterium halobium
Authors: Hildebrand E , Schimz A
Published: November 2016

Stimulation of Halobacterium halobium through its sensory photosystems, PS 370 and PS 565, leads either to a prolonged or to a shortened interval between two reversals of the swimming direction of the cell, the attractant or repellent response. Stimuli are integrated to yield the same response regardless through which photosystem they are given. Simultaneously elicited attractant and repellent signals cancel each other at any time during a reversal interval, even in the period of refractoriness shortly after a reversal, when the cell is insensitive to repellent stimuli. Successively applied stimuli are less completely integrated. The net response depends on the moment of stimulation during the interval, on the sequence of stimuli, and on the delay between them. Integration of successively applied effective stimuli (after refractoriness) is to a great extent explained in terms of a cellular oscillator (A. Schimz and E. Hildebrand, Nature [London] 317:641-643, 1985) which is changed in opposite directions by attractant and repellent signals. Some conclusions on the shape of the oscillator after its disturbance by a stimulus can be made. Integration of signals during refractoriness leads us to postulate an additional step before the oscillator in the sensory pathway. Cancelling of simultaneous opposite signals is thought to proceed at this integrator. It also takes part in the integration of successively evoked signals. At this step signals rapidly decline within 10 ms, and the total life time (at least of repellent signals) does not exceed 1.2 s.
Replication of plasmid RK2 in vitro by a DNA-membrane complex: evidence for initiation of replication and its coupling to transcription and translation
Authors: Kornacki J A , Firshein W
Published: November 2016

The following results with an in vitro replication system utilizing a plasmid RK2 DNA-membrane complex indicate that the essential trfA-encoded replication protein of RK2 is present and active in the complex. (i) A complex extracted from a conditional replication mutant of RK2, which contains a temperature-sensitive mutation in trfA, displayed extensive DNA synthesis at the permissive temperature but little activity at the restrictive temperature. A control wild-type RK2 complex showed no inhibition of DNA synthesis at the restrictive temperature. (ii) Analysis of plasmid-encoded proteins revealed that the trfA-specified replication protein and other proteins which may be involved in the replication and maintenance of RK2 are located physically in the complex. Semiconservative plasmid DNA replication by the DNA-membrane complex was indicated by density shift experiments; DNA synthesized in the presence of a heavy-density precursor banded primarily in a heavier-density area of a neutral CsCl density gradient and consisted mostly of heavy- and light-density single-stranded DNA as determined by alkaline CsCl density gradient centrifugation. Plasmid RK2 DNA replication by the DNA-membrane complex appears to be coupled to transcription and translation as indicated by the following results: the inhibitory effects of chloramphenicol on both DNA and protein synthesis by the complex; the stimulation of replication by components normally required for protein synthesis (tRNA and all the common amino acids); the synthesis of RNA and protein by the complex; and the synthesis of specific RK2-encoded proteins.
Cloning, mapping, and sequencing of plasmid R100 traM and finP genes
Authors: Fee B E , Dempsey W B
Published: November 2016

The fertility control gene finP, the transfer gene traM, and the transfer origin, oriT, of plasmid R100 were isolated on a single 1.2-kilobase EcoRV fragment and were then subcloned as HaeIII fragments. The sequence of the 754-base-pair finP-containing fragment is reported here. In addition to the finP gene, the sequence includes all but two bases of the R100 traM open reading frame and apparently all of the leader mRNA sequence and amino end of the traJ gene of R100. The sequence contains two open reading frames which encode small proteins on the opposite strand from the traM and traJ genes. It also shows two sets of inverted repeats that have the characteristics of transcription terminators. One set is positioned as if it was the traM terminator, and the other set, which is downstream from the first, sits in the middle of the leader mRNA sequence for traJ. On the bottom strand, this inverted repeat has the structure of a rho-independent terminator. Other less-stable inverted repeats overlap this second terminator in the same way as is seen in attenuation sequences, and the two separate small open reading frames on the bottom strand also totally overlap the stem of the rho-independent terminator, suggesting that their translation would cause shifting of termination to the bottom strand homolog of the putative traM terminator. The finP gene product was not identified, but the gene was mapped to the sequence which contains the traJ gene. It either overlaps traJ or is antisense to it.
Effects of salts on the lethality of paraquat
Authors: Kitzler J , Fridovich I
Published: November 2016

Escherichia coli suffered 95 to 100% lethality when exposed to 1.0 mM paraquat for 30 min at 37 degrees C in aerobic nutrient broth medium but did not lose viability when the exposure was done in Vogel Bonner or tryptic soy yeast extract medium. Paraquat was, however, bacteriostatic in all of these media. Salts, added to the nutrient broth medium, protected against the lethality of paraquat, whereas sucrose did not. Salts of divalent cations were much more effective than salts of monovalent cations. Paraquat increases cyanide-resistant respiration by E. coli; salts added before, but not after, the paraquat diminished this effect. 2,4-Dinitrophenol similarly decreased the cyanide-resistant respiration when added before, but not after, the paraquat. The lethality imposed by paraquat correlated with the rate of cyanide-resistant respiration whether this respiration was modulated by varying salt concentration at a fixed concentration of paraquat or by varying paraquat concentration at a fixed concentration of salt. We conclude that salts or 2,4-dinitrophenol interferes with the active uptake of paraquat by E. coli and thus prevents its lethal effect. The salt concentrations found in a number of commonly used microbiological media are sufficient to exert this effect.
Nonrandom cosmid cloning and prophage SP beta homology near the replication terminus of the Bacillus subtilis chromosome
Authors: Rowe D B , Iismaa T P , Wake R G
Published: November 2016

From a library of Bacillus subtilis DNA cloned with the Escherichia coli cosmid vector pHC79, 85 recombinant cosmids containing DNA from near the replication terminus, terC, were identified. The DNA inserts of these cosmids were confined to three regions of a 350-kilobase segment of the chromosome extending from the left end of the SP beta prophage to approximately 75 kilobases on the right of terC. All B. subtilis genes known to reside in this segment, as well as the portion of the SP beta prophage that is expressed early in the lytic cycle of the phage, appeared to be absent from the library. A region of SP beta homology distinct from the prophage and just to the left of terC was identified.
Sustained ethylene production in Agrobacterium-transformed carrot disks caused by expression of the T-DNA tms gene products
Authors: Goodman T C , Montoya A L , Williams S , Chilton M D
Published: November 2016

Agrobacterium-infected carrot disks continually produced elevated levels of ethylene. Ethylene production was mediated by the elevated levels of auxin synthesized in transformed tissues.
Identification of a carotenoid-binding protein in the cytoplasmic membrane from the heterotrophic cyanobacterium Synechocystis sp. strain PCC6714
Authors: Bullerjahn G S , Sherman L A
Published: November 2016

We isolated a carotenoid-binding protein from the cytoplasmic membrane of the cyanobacterium Synechocystis sp. strain PCC6714. The polypeptide demonstrated a characteristic mobility shift when electrophoresed in lithium dodecyl sulfate-polyacrylamide gels. The protein migrated with an apparent molecular mass of 35 kilodaltons when solubilized at 0 degrees C, but after solubilization at 70 degrees C, the protein migrated as a 45-kilodalton species. The carotenoid-binding protein accumulated only in autotrophically grown cells; cytoplasmic membranes prepared from photoheterotrophically grown cells lacked this component.
Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene
Authors: Kawakami B , Kudo T , Narahashi Y , Horikoshi K
Published: November 2016

Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed. Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase. The synthesis of NALase in E. coli carrying these plasmids was constitutive.
Functional relationship among the gene clusters encoding F7(1), F7(2), F9, and F11 fimbriae of human uropathogenic Escherichia coli
Authors: Van Die I , Van Megen I , Zuidweg E , Hoekstra W , De Ree H , Van den Bosch H , Bergmans H
Published: November 2016

The P fimbrial gene clusters encoding the serologically different F7(1), F7(2), F9, and F11 fimbriae were compared functionally. The results show that these gene clusters are closely related.
Expression of luciferases from Vibrio harveyi and Vibrio fischeri in filamentous cyanobacteria
Authors: Schmetterer G , Wolk C P , Elhai J
Published: November 2016

Shuttle vectors that had previously been shown to replicate both in Escherichia coli and in strains of Anabaena spp. were used to transfer the lux genes from Vibrio harveyi and Vibrio fischeri into Anabaena spp. The level of expression of luciferase in the cyanobacteria (up to 7,000 quanta cell-1 s-1) makes these genes good candidates for use as promoter probes during the differentiation of certain cells in a filament into heterocysts.
Translational regulatory signals within the coding region of the bacteriophage lambda cIII gene
Authors: Altuvia S , Oppenheim A B
Published: November 2016

Six independent mutations which enhance the lysogenic response were analyzed. The mutations cause single-base substitutions at three sites within the cIII coding sequence, one of which does not change the amino acid code. The mutations allow for elevated translation of the cIII gene, possibly via changes in the mRNA secondary structure.
Regulatory mutation that controls nif expression and histidine transport in Azospirillum brasilense
Authors: Fischer M , Levy E , Geller T
Published: November 2016

Mutagenesis of Azospirillum brasilense with nitrosoguanidine and selection on ethylenediamine yielded prototrophs which fixed nitrogen in the presence of ammonia. Nitrogenase activity in mutant strains exceeded that of the wild type three- to sixfold. The same mutants were also constitutive for histidine transport. Enzyme activities involved in ammonia assimilation were not affected by the mutation. The data suggest that the mutation occurred at a site which regulates nif and histidine transport functions.
Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1)
Authors: Actis L A , Fish W , Crosa J H , Kellerman K , Ellenberger S R , Hauser F M , Sanders-Loehr J
Published: November 2016

Anguibactin, a siderophore produced by cells of Vibrio anguillarum 775 harboring the pJM1 plasmid, has now been isolated from the supernatants of iron-deficient cultures. This iron-reactive material was purified by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column. The resulting neutral compound produced an ion at m/z 348 in mass spectrometry and contained one sulfur, four oxygen, and four nitrogen atoms as determined by elemental analysis. Its strong UV absorbance and blue fluorescence were suggestive of a phenolic moiety. In colorimetric reactions anguibactin behaved like a catechol. The catechol assignment was supported by the appearance of a new absorption band at 510 nm in the ferric complex and by the appearance of peaks at 1,367, 1,447, 1,469, and 1,538 cm-1 in the resonance Raman spectrum. In addition, the infrared spectrum gave evidence of a secondary amide function, but no free carboxylic acid or hydroxamic acid groups were observed. A third iron-ligating group was suggested by the liberation of three protons during iron binding; mass spectrometry of the resulting material yielded a molecular ion characteristic of a 1:1 complex of ferric anguibactin. The purified anguibactin exhibited specific growth-promoting activity under iron-limiting conditions for a siderophore-deficient mutant of V. anguillarum 775(pJM1). A novel structure for anguibactin was indicated by the failure of a large number of known siderophores and synthetic chelators to yield a similar type of specific cross-feeding in the V. anguillarum bioassay.
Translational control of exported proteins in Escherichia coli
Authors: Hengge-Aronis R , Boos W
Published: November 2016

We recently described the suppression of export of a class of periplasmic proteins of Escherichia coli caused by overproduction of a C-terminal truncated periplasmic enzyme (GlpQ'). This truncated protein was not released into the periplasm but remained attached to the inner membrane and was accessible from the periplasm. The presence of GlpQ' in the membrane strongly reduced the appearance in the periplasm of some periplasmic proteins, including the maltose-binding protein (MBP), but did not affect outer membrane proteins, including the lambda receptor (LamB) (R. Hengge and W. Boos, J. Bacteriol., 162:972-978, 1985). To investigate this phenomenon further we examined the fate of MBP in comparison with the outer membrane protein LamB. We found that not only localization but also synthesis of MBP was impaired, indicating a coupling of translation and export. Synthesis and secretion of LamB were not affected. The possibility that this influence was exerted via the level of cyclic AMP could be excluded. Synthesis of MBP with altered signal sequences was also reduced, demonstrating that export-defective MBP which ultimately remains in the cytoplasm abortively enters the export pathway. When GlpQ' was expressed in a secA51(Ts) strain, the inhibition of MBP synthesis caused by GlpQ' was dominant over the precursor accumulation usually caused by secA51(Ts) at 41 degrees C. Therefore, GlpQ' acts before or at the level of recognition by SecA. For LamB the usual secA51(Ts) phenotype was observed. We propose a mechanism by which GlpQ' blocks an yet unknown membrane protein, the function of which is to couple translation and export of a subclass of periplasmic proteins.
Release of cell-free ice nuclei by Erwinia herbicola
Authors: Phelps P , Giddings T H , Prochoda M , Fall R
Published: November 2016

Several ice-nucleating bacterial strains, including Erwinia herbicola, Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for their ability to shed ice nuclei into the growth medium. Only E. herbicola isolates shed cell-free ice nuclei active at -2 to -10 degrees C. These cell-free nuclei exhibited a freezing spectrum similar to that of ice nuclei found on whole cells, both above and below -5 degrees C. Partially purified cell-free nuclei were examined by density gradient centrifugation, chemical and enzymatic probes, and electron microscopy. Ice-nucleating activity in these cell-free preparations was associated with outer membrane vesicles shed by cells and was sensitive to protein-modifying reagents.
Chromosomal insertions of Tn917 in Bacillus subtilis
Authors: Vandeyar M A , Zahler S A
Published: November 2016

We describe 46 insertions of the Streptococcus faecalis transposon Tn917 into the chromosome of Bacillus subtilis. These insertion mutations were mapped genetically. Some caused auxotrophic requirements, and others were cryptic. These insertions were scattered around the B. subtilis chromosome. The mutant strains were useful in several ways for mapping and cloning B. subtilis genes and were added to the Bacillus Genetic Stock Center collection. Among the auxotrophic markers were a new serine auxotrophy and deletion-insertions that caused auxotrophy in one case for homoserine and threonine, in another case for uracil and either cysteine or methionine, and in a third case for leucine, isoleucine, and valine.
Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome
Authors: Takagi M , Kawai S , Chang M C , Shibuya I , Yano K
Published: November 2016

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.
Molecular analysis of the promoter operator region of the Escherichia coli K-12 tyrP gene
Authors: Kasian P A , Davidson B E , Pittard J
Published: November 2016

The nucleotide sequence of the tyrP promoter region from Escherichia coli has been determined. Two TYR R boxes have been identified, and one of these was shown to overlap the -35 region of a major tyrP promoter (p1). S1 nuclease mapping of in vivo transcripts revealed that transcription from p1 is stimulated by phenylalanine and to a lesser extent by leucine. The demonstration that mutants in which TyrR-tyrosine-mediated repression of tyrP has been abolished have single base changes in the TYR R box which overlaps p1 suggests that TyrR-tyrosine-mediated repression of tyrP also involves p1. TyrR-independent stimulation of tyrP expression by Casamino Acids involves a second promoter 140 bases upstream of p1. There are no TYR R boxes in this region. The sequences of 10 TYR R boxes preceding the genes tyrP, tyrR, and aroG and the operons aroF tyrA and aroL aroM are compared and discussed.
Relationship of shape to initiation of new sites of envelope growth in Streptococcus faecium cells treated with beta-lactam antibiotics
Authors: Higgins M L , Ferrero M , Daneo-Moore L
Published: November 2016

Exponential-phase cells of Streptococcus faecium were treated with concentrations of ampicillin and cephalothin which, over 60 min, had little effect on increase in culture mass but resulted in about a 65% inhibition of increase in cell numbers. The resulting drug-treated cells underwent about a doubling in cell mass and volume above that of the untreated cells. The newly divided cells produced in the presence of drugs were shown to be due to the division of central or primary sites of envelope growth present at the time of treatment. Sites that were newly initiated (secondary sites) at the time of treatment or sites initiated after treatment did not divide but enlarged in length and girth to give abnormally large cells. Although the increase in average total volume was the same after each interval of treatment with ampicillin and cephalothin, the primary growth sites of the cephalothin-treated cells grew somewhat more slowly, and their secondary sites grew somewhat more quickly, than did those of the ampicillin-treated cells. Cephalothin-treated cells initiated secondary sites at a rate similar to that of the untreated cells, whereas the ampicillin-treated cells exhibited reduced rates of secondary site initiation. Two models are presented that account for these results.
Overproduction and assay of Pseudomonas aeruginosa phosphomannose isomerase
Authors: Gill J F , Deretic V , Chakrabarty A M
Published: November 2016

Phosphomannose isomerase activity was undetectable in extracts of mucoid (alginate-producing) Pseudomonas aeruginosa. When a P. aeruginosa gene previously shown to complement an alginate-negative mutant was overexpressed under the control of the tac promoter in the broad-host-range controlled-expression vector pMMB22, phosphomannose isomerase activity could be measured in extracts of P. aeruginosa and in a manA (phosphomannose isomerase-negative) mutant of Escherichia coli. P. aeruginosa extracts containing induced levels of enzyme were shown to interconvert fructose 6-phosphate and mannose 6-phosphate. A 56,000-dalton polypeptide was visualized on sodium dodecyl sulfate-polyacrylamide gels after induction in both hosts. When RNA-DNA dot- blot hybridization analysis was used, transcription of algA, the gene coding for P. aeruginosa phosphomannose isomerase, was not measurable from the chromosomes of either mucoid or nonmucoid P. aeruginosa. However, a high level of algA transcription was detected after expression of algA under tac promoter control in pMMB22.
crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli
Authors: Cossart P , Groisman E A , Serre M C , Casadaban M J , Gicquel-Sanzey B
Published: November 2016

The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.