bluuebunny/crossref_metadata_2025 huggingface dataset:
A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae
Authors: Schmidheini T , Sperisen P , Paravicini G , Hütter R , Braus G
Published: November 2016

The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.
Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector
Authors: Shoemaker N B , Barber R D , Salyers A A
Published: November 2016

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.
Changes in gene expression during nitrogen starvation in Anabaena variabilis ATCC 29413
Authors: Wealand J L , Myers J A , Hirschberg R
Published: November 2016

When the filamentous, nitrogen-fixing cyanobacterium Anabaena variabilis ATCC 29413 was subjected to nitrogen starvation under aerobic conditions, a complex series of events was initiated which resulted in heterocyst formation and derepression of the ability to fix dinitrogen. Using DNA-RNA hybridization techniques, we monitored the expression of several genes during nitrogen starvation and correlated changes in the mRNA levels with changes in enzyme activity, protein levels, and morphology. Nitrogenase mRNA was first observed after about 8.5 h of nitrogen starvation, as was nitrogenase activity. Late proheterocysts were present at that time. The level of nitrogenase mRNA increased for 5 to 6 h and then leveled off. Phycocyanin and allophycocyanin mRNA levels decreased rapidly within 1 h of nitrogen starvation; the levels increased later, as nitrogen starvation was alleviated, first by protein breakdown and then by nitrogen fixation. The average half-life of A. variabilis mRNA was determined by pulse-labeling techniques to be 16 to 18 min. Hybridization analysis showed that cpc and apc mRNAs also had half-lives of 16 to 18 min; the half-lives were not significantly different under nitrogen starvation conditions. Our results support the idea that the changes induced by nitrogen starvation are primarily the result of transcriptional regulation.
Activation of formylmethanofuran synthesis in cell extracts of Methanobacterium thermoautotrophicum
Authors: Bobik T A , Wolfe R S
Published: November 2016

In cell extracts of Methanobacterium thermoautotrophicum, formylmethanofuran (formyl-MFR) synthesis (an essential CO2 fixation reaction that is an early step in CO2 reduction to methane) is subject to a complex activation that involves a heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine O3-phosphate (CoM-S-S-HTP). In this paper we report that titanium(III) citrate, a low-potential reducing agent, stimulated CO2 reduction to methane and activated formyl-MFR synthesis in cell extracts. Titanium(III) citrate functioned as the sole source of electrons for formyl-MFR synthesis and enabled this reaction to occur independently of CoM-S-S-HTP. In addition, CoM-S-S-HTP was found to activate an unknown electron carrier that reduced metronidazole. The activation of formyl-MFR synthesis by CoM-S-S-HTP may involve the activation of a low-potential electron carrier.
Identification and characterization of catA, a mutation causing catalase deficiency in Dictyostelium discoideum
Authors: Madigan S J , Katz E R
Published: November 2016

Various strains of Dictyostelium discoideum were assayed for catalase activity. We were able to demonstrate the presence of catalase in lysates of all strains tested except one, ts12m. Lysates of this strain did not show any detectable level of catalase. The increased sensitivity of intact ts12m amoebae to hydrogen peroxide was consistent with the catalase deficiency. We followed catalase activity through a developmental time course for the wild type (Ddb) and were able to show its presence throughout development. No catalase activity was detected from ts12m in any stage of development. The growth and development of this acatalasemic strain of D. discoideum was no different than those of the wild type. The mutation, catA, was assigned to linkage group II.
Changes in diadenosine tetraphosphate levels in Physarum polycephalum with different oxygen concentrations
Authors: Garrison P N , Mathis S A , Barnes L D
Published: November 2016

Cellular levels of diadenosine tetraphosphate (Ap4A) were measured, by a specific high-pressure liquid chromatography method, in microplasmodia of Physarum polycephalum subjected to different degrees of hypoxia, hyperoxia, and treatment with H2O2. Ap4A levels increased three- to sevenfold under anaerobic conditions, and the microplasmodia remained viable after such treatment. Elevated levels of Ap4A returned to the basal level within 5 to 10 min upon reoxygenation of the microplasmodia. The increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in fed, log-phase microplasmodia. The maximal increase measured in log-phase microplasmodia was twofold. No significant changes in Ap4A levels occurred in microplasmodia subjected to mild hypoxia, hyperoxia, or treatment with 1 mM H2O2. These results indicate that in P. polycephalum, Ap4A may function in the metabolic response to anaerobic conditions rather than in the response to oxidative stress.
Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro
Authors: Johnson C , Chandrasekhar G N , Georgopoulos C
Published: November 2016

Previous studies have demonstrated that the Escherichia coli dnaK and grpE genes code for heat shock proteins. Both the Dnak and GrpE proteins are necessary for bacteriophage lambda DNA replication and for E. coli growth at all temperatures. Through a series of genetic and biochemical experiments, we have shown that these heat shock proteins functionally interact both in vivo and in vitro. The genetic evidence is based on the isolation of mutations in the dnaK gene, such as dnaK9 and dnaK90, which suppress the Tr- phenotype of bacteria carrying the grpE280 mutation. Coimmunoprecipitation of DnaK+ and GrpE+ proteins from cell lysates with anti-DnaK antibodies demonstrated their interaction in vitro. In addition, the DnaK756 and GrpE280 mutant proteins did not coimmunoprecipitate efficiently with the GrpE+ and DnaK+ proteins, respectively, suggesting that interaction between the DnaK and GrpE proteins is necessary for E. coli growth, at least at temperatures above 43 degrees C. Using this assay, we found that one of the dnaK suppressor mutations, dnaK9, reinstated a protein-protein interaction between the suppressor DnaK9 and GrpE280 proteins.
Nisin, a peptide antibiotic: cloning and sequencing of the nisA gene and posttranslational processing of its peptide product
Authors: Kaletta C , Entian K D
Published: November 2016

Nisin produced by Streptococcus lactis is used as a food preservative and is the most important member of a group of antibiotics containing lanthionine bridges. To understand the genetic basis of these so-called lantibiotics (Schnell et al., Nature 333:276-278, 1988), we characterized the nisin structural gene, nisA, which is located on a plasmid and codes for a 57-amino-acid prepeptide. The prepeptide is processed posttranslationally to the pentacyclic antibiotic. Although nisin and the recently elucidated lantibiotic epidermin from Staphylococcus epidermidis are produced by different organisms, their gene organization is identical. As with epidermin, the nisin propeptide corresponds to the C-terminus of the prepeptide. The N-terminus of the prepeptide is cleaved at a characteristic splice site (Pro--2 Arg--1 Ile-+1). Remarkably, the N-terminus of prenisin shares 70% similarity with preepidermin, although the propeptide sequences are distinctly different. The structural similarities between these two lantibiotics are consistent with the fact that there is a common mechanism of biosynthesis of these lanthionine-containing antibiotics.
Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan
Authors: Cangelosi G A , Martinetti G , Leigh J A , Lee C C , Theines C , Nester E W
Published: November 2016

Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic beta-1,2-glucan. Whereas chvB is required for beta-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral beta-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble beta-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for beta-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.
Cyclic-AMP-dependent switch in initiation of transcription from the two promoters of the Escherichia coli gal operon: identification and assay of 5'-triphosphate ends of mRNA by GTP:RNA guanyltransferase
Authors: Irani M , Musso R , Adhya S
Published: November 2016

We have studied the initiation of transcription of the gal operon in Escherichia coli (i) by analyzing the 5'-triphosphate ends and (ii) by measuring the level of promoter-proximal gal mRNA made in vivo. The 5' termini were identified and quantified by capping with GTP:mRNA guanyltransferase, and the mRNA levels were determined by hybridization of pulse-labeled [32P]RNA with a specific DNA probe. Our results conclusively demonstrate the in vivo activities of two promoters, P1 and P2, with separate initiation sites (S1 and S2) as suggested before from in vitro and in vivo experiments (S. Adhya and W. Miller, Nature [London] 279:492-494, 1979; R. E. Musso, R. DiLauro, S. Adhya, and B. de Crombrugghe, Cell 12:847-854, 1977). We have also studied the effect of cyclic AMP (cAMP) on in vivo gal transcription and found that whereas total gal transcription remains largely unchanged, the relative proportions of the S1 and S2 mRNAs are influenced by the level of cAMP in the cell. In strains devoid of cAMP (cya), transcription initiates equally at S1 and S2; in cAMP-proficient cells (cya+), the S1 initiation increases twofold with a concomitant decrease in S2 initiation. Addition of a saturating amount of exogenous cAMP to cya mutant cells results in a relatively larger switch from S2 to S1. Our results clearly show that while cAMP is an inhibitor of S2, it is not an absolute requirement for transcription initiation at S1, but only acts to increase low-level transcription from the P1 promoter. Using these approaches, we have also studied gal promoter mutants (P211, P18, and P35) which show altered behavior in transcription initiations and in response to cAMP. On the basis of these results, we have discussed models by which transcription initiates at the two overlapping gal promoters (P1 and P2) and discussed how cAMP level modulates the switch between them.
Extent of the DNA sequence required in integration of staphylococcal bacteriophage L54a
Authors: Lee C Y , Buranen S L
Published: November 2016

We characterized the minimum length of the DNA sequence of the attachment sites involved in the integrative recombination of staphylococcal bacteriophage L54a. A DNA fragment carrying the functional viral attachment site (attP) or the bacterial attachment site (attB) was sequentially trimmed, recloned, and tested for integrative recombination in vivo. The size of the functional attP site was at least 228 base pairs (bp) but no more than 235 bp. The left endpoint of the attP site was located to between positions -142 and -140, whereas the right endpoint was located to between positions +86 and +93 with respect to the center of the core sequence. The attB site was located to within a 27-bp sequence, from position -15 to +12, which included the 18-bp core sequence.
Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation
Authors: de Bruijn F J , Rossbach S , Schneider M , Ratet P , Messmer S , Szeto W W , Ausubel F M , Schell J
Published: November 2016

We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT). The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation. The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII). The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E. coli strain. The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons. glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+). A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules. glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules. glnII and glnT, but not glnA, expression in R. meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.
Cloning and sequence analysis of flaA, a gene encoding a Spirochaeta aurantia flagellar filament surface antigen
Authors: Brahamsha B , Greenberg E P
Published: November 2016

Spirochaeta aurantia DNA that coded for an antigenic determinant of the flagellin associated with the filament surface of the periplasmic flagella was isolated. When expressed in Escherichia coli, the antigenic polypeptide had an apparent molecular weight of 37,000. Sequence analysis of the antigen-encoding DNA revealed the presence of an open reading frame that determined a polypeptide with a predicted molecular weight of 31,241. This polypeptide showed a region of identity with the N-amino-terminal region of the 39,000- and 37,000-dalton flagellins of the distantly related spirochetes Treponema phagedenis and Treponema pallidum, respectively (S. J. Norris, N. W. Charon, R. G. Cook, M. D. Fuentes, and R. J. Limberger, J. Bacteriol. 170:4072-4082, 1988). The region of identity in the deduced S. aurantia polypeptide was preceded by a possible signal sequence and signal peptidase cleavage site.
Molecular cloning and expression in Escherichia coli of the Bacillus licheniformis bacitracin synthetase 2 gene
Authors: Ishihara H , Hara N , Iwabuchi T
Published: November 2016

The structural genes for the entire bacitracin synthetase 2 (component II) and for a part of the putative bacitracin synthetase 3 (component III) from Bacillus licheniformis ATCC 10716 were cloned and expressed in Escherichia coli. A cosmid library of B. licheniformis DNA was constructed. The library was screened for the ability to produce bacitracin synthetase by in situ immunoassay using anti-bacitracin synthetase antiserum. A positive clone designated B-15, which has a recombinant plasmid carrying about a 32-kilobase insert of B. licheniformis DNA, was further characterized. Analysis of crude cell extract from B-15 by polyacrylamide gel electrophoresis and Western blotting (immunoblotting) showed that the extract contains two immunoreactant proteins with high molecular weight. One band with a molecular weight of about 240,000 comigrates with bacitracin synthetase 2; the other band is a protein with a molecular weight of about 300,000. Partial purification of the gene products encoded by the recombinant plasmid by gel filtration and hydroxyapatite column chromatography revealed that one gene product catalyzes L-lysine- and L-ornithine-dependent ATP-PPi exchange reactions which are characteristic of bacitracin synthetase 2, and the other product catalyzes L-isoleucine-, L-leucine, L-valine-, and L-histidine-dependent ATP-PPi exchange activities, suggesting the activities of a part of bacitracin synthetase 3. Subcloning experiments indicated that the structural gene for bacitracin synthetase 2 is located near the middle of the insert.
Rhizobium meliloti regulatory gene fixJ activates transcription of R. meliloti nifA and fixK genes in Escherichia coli
Authors: Hertig C , Li R Y , Louarn A M , Garnerone A M , David M , Batut J , Kahn D , Boistard P
Published: November 2016

When present in Escherichia coli on the multicopy expression vector pUC19, a Rhizobium meliloti regulatory gene, fixJ, belonging to a two-component regulatory system, activated the expression of two R. meliloti symbiotic genes, nifA and fixK. Primer extension by reverse transcription showed that FixJ stimulates nifA expression in E. coli by activating pnifA.
Mutation of Bacillus firmus OF4 to duramycin resistance results in substantial replacement of membrane lipid phosphatidylethanolamine by its plasmalogen form
Authors: Clejan S , Guffanti A A , Cohen M A , Krulwich T A
Published: November 2016

Mutant strains of alkalophilic Bacillus firmus OF4 that were selected for resistance to duramycin had greatly reduced levels of membrane diacylphosphatidylethanolamine, as had been found in studies of such mutants of Bacillus subtilis. In the B. firmus strains, however, substantial levels of plasmenylethanolamine were found. This is an unusual membrane component for an aerobic eubacterium, but the presence of trace amounts even in the wild type was confirmed in experiments with 32Pi-labeled growth medium. The membrane lipid composition of the duramycin-resistant strains had several other changes that also left alkalophilic growth unimpaired.
13N isotope studies of glutamine assimilation pathways in Neurospora crassa
Authors: Calderón J , Cooper A J , Gelbard A S , Mora J
Published: November 2016

L-[amide-13N]glutamine in Neurospora crassa is metabolized to [13N]glutamate by glutamate synthase and to [13N]ammonium by the glutamine transaminase-omega-amidase pathway. The [13N]ammonium released is assimilated by glutamate dehydrogenase and glutamine synthetase, confirming the operation of a glutamine cycle. Most of the nitrogen is retained during cycling between glutamate and glutamine.
Novel change in the carbohydrate portion of Myxococcus xanthus lipopolysaccharide during development
Authors: Panasenko S M , Jann B , Jann K
Published: November 2016

We have examined the alterations in lipopolysaccharide during aggregation and early development in Myxococcus xanthus. The lipopolysaccharide was isolated and characterized from cells developing on agar during glycerol induction and vegetative growth. A methylated amino sugar was identified as 6-O-methylgalactosamine by gas-liquid chromatography-mass spectrometry. This novel sugar was enriched in cells developing on agar.
Amplification of DNA at a prophage attachment site in Haemophilus influenzae
Authors: Kauc L , Goodgal S H
Published: November 2016

The Escherichia coli plasmids pBR322 and pBR327 can be taken up by Haemophilus influenzae but do not replicate in this organism; however, integration of pBR into the H. influenzae chromosome was achieved by ligation to a fragment of the Haemophilus phage S2 that carried a phage attachment site (attP). Once these sequences were integrated, they could serve as sites of recombination and amplification for homologous (pBR or phage) DNA. Amplification appeared to occur in one of two prophage sites (attB) present in the H. influenzae chromosome. The extent of amplification was different in different cells and reflected the ability of these sequences to undergo rearrangement leading to the formation of a DNA ladder. The ladder was obtained by treatment of DNA with restriction enzymes that cut outside of the inserted DNA, i.e., did not cut in the repeat sequence, and represented different numbers of repeat elements. Reversed-field gel electrophoresis was instrumental in resolving amplified structures. Inasmuch as single-cell isolates gave rise to the same ladder structure, it was assumed that amplification was under regulatory control and that it reproduced the same equilibrium of repeat structures. Transformation of E. coli with the amplified H. influenzae DNA resulted in precise excision and replication of the original monomeric plasmids. This excision was independent of the recA and recBC genes.
Changes in polypeptide composition of Synechocystis sp. strain 6308 phycobilisomes induced by nitrogen starvation
Authors: Duke C S , Cezeaux A , Allen M M
Published: November 2016

Phycobilisomes isolated from actively growing Synechocystis sp. strain 6308 (ATCC 27150) consist of 12 polypeptides ranging in molecular mass from 11.5 to 95 kilodaltons. The phycobilisome anchor and linker polypeptides are glycosylated. Nitrogen starvation causes the progressive loss of phycocyanin and allophycocyanin subunits with molecular masses between 16 and 20 kilodaltons and of two linker polypeptides with molecular masses of 27 and 33 kilodaltons. Nitrogen starvation also leads to enrichment of four additional polypeptides with molecular masses of 46, 53, 57, and 61 kilodaltons and a transient enrichment of 35- and 41-kilodalton polypeptides in isolated phycobilisomes. The 57-kilodalton additional polypeptide was identified by immunoblotting as the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proteins with the same molecular weights as the additional polypeptides were also coisolated with the 12 phycobilisome polypeptides in the supernatant of nitrogen-replete Synechocystis thylakoid membranes extracted in high-ionic-strength buffer and washed with deionized water. These observations suggest that the additional polypeptides in phycobilisomes from nitrogen-starved cells may be soluble or loosely bound membrane proteins which associate with phycobilisomes. The composition and degree of association of phycobilisomes with soluble and adjacent membrane polypeptides appear to be highly dynamic and specifically regulated by nitrogen availability. Possible mechanisms for variation in the strength of association between phycobilisomes and other polypeptides are suggested.
Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus
Authors: Ross T K , Achberger E C , Braymer H D
Published: November 2016

The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.
Effect of cell cycle stages on the central density of Enterococcus faecium ATCC 9790
Authors: Bourbeau P , Dicker D , Higgins M L , Daneo-Moore L
Published: November 2016

Cultures of Enterococcus faecium growing at various rates were examined for timing of cell division cycle events by using the method of residual divisions and a morphological analysis. Both methods gave essentially the same timing for the onset of D1 (completion of chromosome replication) and of D2 (completion of septation). Frequencies of cells exhibiting a phase-reversed center in bovine serum albumin at various growth rates were determined. The data fit a model in which rapidly growing cells increase in refractive index (which is assumed to represent central density) at completion of the chromosome replication cycle involved in the ongoing division, whereas slowly growing cultures increase in central density at the time of completion of septation. There was no correlation between the timing of increase in central density and the timing of initiation of new sites of surface growth.
Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence
Authors: Margolin W , Rao G , Howe M M
Published: November 2016

Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein. Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter. S1 nuclease protection experiments were used to map RNA 5' ends in the three new regions. Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome. Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene. A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli sigma 70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E. coli -35 consensus. We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity. Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent beta-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation. As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage. The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.
Isolation of cell surface antigen mutants of Myxococcus xanthus by use of monoclonal antibodies
Authors: Fink J M , Kalos M , Zissler J F
Published: November 2016

Monoclonal antibodies (MAbs) with affinities for molecules on the cell surface of the procaryote Myxococcus xanthus were used in a screening strategy for the isolation of mutants lacking particular cell surface molecules. From a large library of independent mutants created by Tn5 transposon mutagenesis, mutants were isolated which lacked reactivities with MAb 1604 (a MAb specific for a cell surface protein) and MAbs 2600, 1733, 1514, 1412, and 783 (MAbs specific for carbohydrate epitopes on the O antigen of lipopolysaccharide [LPS]). The defect in antibody recognition was shown by genetic crosses and DNA hybridization experiments to be caused by the Tn5 transposon acting as a mutation at a single locus. Quantitative enzyme-linked immunosorbent assays showed that particular mutant strains had no detectable affinity for the specific MAb probe. LPS mutants were resistant to myxophage Mx8, and this provided a selection method for isolating a large number of new LPS mutants. A class of Mx8-resistant mutants lacked reactivity with MAb 1514 and therefore was defective in the O antigen of LPS. A class of Mx1-resistant mutants lacked reactivity with MAb 2254, a MAb specific for a carbohydrate epitope on the core of LPS. A comparison of MAb binding to different mutant strains revealed a principle for mapping epitopes and showed that MAbs 1514 and 2254 recognize side-chain carbohydrates rather than backbone carbohydrates within the LPS molecule.
Transcriptional and posttranscriptional control of the Bacillus subtilis succinate dehydrogenase operon
Authors: Melin L , Rutberg L , von Gabain A
Published: November 2016

The amount of succinate dehydrogenase (SDH) in Bacillus subtilis varies with growth conditions. In this work we studied the steady-state level and the rate of decay of B. subtilis sdh mRNA under different growth conditions. In exponentially growing cells, the steady-state level of sdh mRNA was severalfold lower when glucose was present compared with growth without glucose, whereas the rate of decay of sdh mRNA was the same with and without glucose. Thus, glucose repression seems to act by decreasing sdh mRNA synthesis. When the bacteria entered the stationary phase, the steady-state level of sdh mRNA dropped about sixfold. At the same time, sdh mRNA half-life decreased from 2.6 to 0.4 min. This result indicates that transcription of the sdh operon is initiated at the same rate in exponentially growing and in stationary-phase cells. The start point of the sdh transcripts, as measured by primer extension, was the same under all conditions studied, suggesting that the sdh operon is solely controlled by the previously identified sigma 43-like promoter. The increase of SDH activity in stationary phase may be explained by reduced dilution of the SDH proteins as a result of the retarded growth rate. We suggest that enhanced degradation of the sdh transcript is a means by which the bacteria adjust expression to the demands of stationary phase.
Cell-associated pullulanase from Bacteroides thetaiotaomicron: cloning, characterization, and insertional mutagenesis to determine role in pullulan utilization
Authors: Smith K A , Salyers A A
Published: November 2016

We have cloned a pullulanase gene from Bacteroides thetaiotaomicron. The pullulanase expressed from this clone in Escherichia coli was cell associated and soluble and had a molecular mass of 72 kilodaltons by gel filtration. Maxicell analysis of proteins coded by the cloned insert showed that a 71.6- to 73.2-kilodalton doublet was associated with pullulanase activity. Thus, the pullulanase is probably a monomer. The cloned pullulanase produced maltotriose as an end product of pullulan digestion. In B. thetaiotaomicron the pullulanase activity was cell associated. Approximately 80% of the activity was soluble, and 16 to 18% was membrane associated. The molecular mass of the soluble pullulanase was 77 kilodaltons by gel filtration. To determine whether the cloned pullulanase gene was essential for pullulan utilization, we used directed insertional mutagenesis to inactivate the B. thetaiotaomicron pullulanase gene. The pullulanase specific activity of the mutant was approximately 45% of that of wild-type B. thetaiotaomicron. However, the pullulanase-negative insertional mutant 95-1 was still able to grow on pullulan at a rate similar to that of wild-type B. thetaiotaomicron. Thus, there must be a second pullulanase in B. thetaiotaomicron.
Formation of supercoiling domains in plasmid pBR322
Authors: Lodge J K , Kazic T , Berg D E
Published: November 2016

Twin domains of positive and negative supercoiling are thought to form in DNA molecules whenever free rotation of a transcription complex around the DNA helix is impeded. Evidence for these domains has come from findings with Escherichia coli strains that are deficient in DNA topoisomerase I (top mutants) or that have been treated with DNA gyrase inhibitors. Plasmid pBR322 is highly supercoiled in these strains, whereas some of its deletion derivatives are not. The studies of pBR322 derivatives presented here show that high negative supercoiling in top strains requires translation as well as transcription of the first 98 codons of the tet gene and does not require the divergently transcribed amp gene. The N-terminal region of the TetA protein is thought to insert into the inner membrane. Our results favor models in which supercoiling domains are created when DNA segments are anchored to a large cellular structure via coupled transcription, translation, and membrane insertion of a nascent protein.
Major outer membrane proteins unique to reproductive cells of Hyphomonas jannaschiana
Authors: Shen N , Dagasan L , Sledjeski D , Weiner R M
Published: November 2016

Separation on the basis of molecular weight resolved three proteins specific to the swarmer cell of Hyphomonas jannaschiana. In the reproductive cell, 4 major proteins were identified as cytoplasmic and 10 were identified as envelope. Of these envelope proteins, one was common to both the inner and outer membranes, four were common to the inner membrane, and five were common to the outer membrane. Four of these outer membrane proteins were specific to the reproductive cell, and two of these proteins, with apparent molecular weights of 116,000 and 29,000, constituted 19% of the total cell protein and 54% of the outer membrane protein.
A cloned regulatory gene of Streptomyces lividans can suppress the pigment deficiency phenotype of different developmental mutants
Authors: Stein D , Cohen S N
Published: November 2016

We report here the cloning of a Streptomyces lividans gene that when introduced on a multicopy plasmid vector reversed the pigment deficiency phenotype of several distinct mutants blocked in development, pigment production, or both. Although this gene was shown by restriction enzyme analysis to be similar to a previously cloned afsB-complementing gene of Streptomyces coelicolor, we show that it does not correspond to the S. coelicolor chromosomal locus designated afsB. Thus, the cloned locus, which we propose to rename afsR, appears to complement the AfsB- phenotype by pleiotropic regulatory effects.
Plasmid structural instability associated with pC194 replication functions
Authors: Ballester S , Lopez P , Espinosa M , Alonso J C , Lacks S A
Published: November 2016

The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When gram-positive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S.D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested. It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.
Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide
Authors: Puziss J W , Fikes J D , Bassford P J
Published: November 2016

Oligonucleotide-directed mutagenesis was employed to investigate the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the protein export process. The three basic residues residing at the amino terminus of the signal peptide were systematically substituted with neutral or acidic residues, decreasing the net charge in a stepwise fashion from +3 to -3. It was found that a net positive charge was not absolutely required for MBP export to the periplasm. However, export was most rapid and efficient when the signal peptide retained at least a single basic residue and a net charge of +1. The nature of the adjacent hydrophobic core helped to determine the effect of charge changes in the hydrophilic segment on MBP export, which suggested that these two regions of the signal peptide do not have totally distinct functions. Although the stepwise decrease in net charge of the signal peptide also resulted in a progressive decrease in the level of MBP synthesis, the data do not readily support a model in which MBP synthesis and export are obligately coupled events. The export defect resulting from alterations in the hydrophilic segment was partially suppressed in strains harboring certain prl alleles but not in strains harboring prlA alleles that are highly efficient suppressors of signal sequence mutations that alter the hydrophobic core.
Isolation, characterization, and biological activity of the Methanococcus thermolithotrophicus ferredoxin
Authors: Hatchikian E C , Fardeau M L , Bruschi M , Belaich J P , Chapman A , Cammack R
Published: November 2016

A ferredoxin has been isolated from the thermophilic methanogen Methanococcus thermolithotrophicus. The native protein was a monomer exhibiting a molecular weight of 7,262, calculated from the amino acid composition. Its absorption spectrum had two maxima at 390 and 283 nm, with an absorbance ratio A390/A283 of 0.79. The absorption at 390 nm (E = 29 mM-1 cm-1) and the content of iron of the protein are in agreement with the presence of two 4Fe-4S clusters in M. thermolithotrophicus ferredoxin. Its amino acid composition showed the presence of eight cysteine residues, which is the required number of cysteines for the binding of two 4Fe-4S clusters. The protein was characterized by the lack of histidine, arginine, and leucine and a high content of valine. It was unusually stable to high temperatures but not to oxygen. The ESR spectrum of the protein in the oxidized state showed a minor signal at g = 2.01, corresponding to an oxidized 3Fe-4S cluster. The protein, which was difficult to reduce with dithionite or reduced mediators, exhibited in its reduced state a spectrum typical of two interacting reduced 4Fe-4S clusters. M. thermolithotrophicus ferredoxin functioned as an electron acceptor for the CO dehydrogenase complex with an extract free of ferredoxin. No reaction was detected with F420 or hydrogenase.
New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis
Authors: Dutreix M , Moreau P L , Bailone A , Galibert F , Battista J R , Walker G C , Devoret R
Published: November 2016

To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.
Size and physical map of the chromosome of Haemophilus influenzae
Authors: Kauc L , Mitchell M , Goodgal S H
Published: November 2016

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.
Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I
Authors: Bates H , Randall S K , Rayssiguier C , Bridges B A , Goodman M F , Radman M
Published: November 2016

The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process. The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type. The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type. The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation. The mutator activity therefore appears to reflect constitutive SOS induction. Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome. The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele. This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria. An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair. Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present.