bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic evidence for interaction of sigma E with the spoIIID promoter in Bacillus subtilis
Authors: Tatti K M , Jones C H , Moran C P
Published: November 2016

During sporulation in Bacillus subtilis, new RNA polymerase sigma factors are produced. These sigma factors direct the transcription of genes that are required for this cellular differentiation. In order to determine the role of each sigma factor in this process, it is necessary to know which promoters are recognized by each sigma factor. The spoIIID gene product plays an important role in the establishment of mother cell-specific gene expression during sporulation. We found that substitution of an alanine at position 124 of the sporulation-specific sigma factor sigma E suppressed the effect of a single-base-pair transition at position -13 of the spoIIID promoter. This alanine substitution in sigma E did not suppress the effect of a transversion at position -12 of the spoIIID promoter. The allele specificity of the interaction between sigma E and the spoIIID promoter is strong evidence that sigma E directs transcription from the spoIIID promoter during sporulation. Position 124 in sigma E is located within a region that is highly conserved among the regions in other sigma factors that probably interact with the -10 regions of their cognate promoters.
Molecular analysis of Bacillus subtilis ada mutants deficient in the adaptive response to simple alkylating agents
Authors: Morohoshi F , Hayashi K , Munakata N
Published: November 2016

Previously, we isolated and characterized six Bacillus subtilis ada mutants that were hypersensitive to methylnitroso compounds and deficient in the adaptive response to alkylation. Cloning of the DNA complementing the defects revealed the presence of an ada operon consisting of two tandem and partially overlapping genes, adaA and adaB. The two genes encoded proteins with methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase activities, respectively. To locate the six mutations, the ada operon was divided into five overlapping regions of about 350 bp. The fragments of each region were amplified by polymerase chain reaction and analyzed by gel electrophoresis to detect single-strand conformation polymorphism. Nucleotide sequences of the fragments exhibiting mobility shifts were determined. Three of the mutants carried sequence alterations in the adaA gene: the adaA1 and adaA2 mutants had a one-base deletion and insertion, respectively, and the adaA5 mutant had a substitution of two consecutive bases causing changes of two amino acid residues next to the presumptive alkyl-accepting Cys-85 residue. Three mutants carried sequence alterations in the adaB gene: the adaB3 mutant contained a rearrangement, the adaB6 mutant contained a base substitution causing a change of the presumptive alkyl-accepting Cys-141 to Tyr, and the adaB4 mutant contained a base substitution changing Leu-167 to Pro. The adaB mutants produced ada transcripts upon treatment with low doses of alkylating agents, whereas the adaA mutant did not. We conclude that the AdaA protein functions as the transcriptional activator of this operon, while the AdaB protein specializes in repair of alkylated residues in DNA.
Ribosome hopping and translational frameshifting are inadequate alternatives to translational attenuation in cat-86 regulation
Authors: Rogers E J , Ambulos N P , Lovett P S
Published: November 2016

The induction of cat-86 by chloramphenicol has been proposed to follow the translational attenuation model. In the absence of inducer, the cat-86 gene is transcribed but remains phenotypically unexpressed because the transcripts sequester the ribosome binding site for the cat coding sequence in a stable stem-loop structure, preventing translation initiation. The translational attenuation model proposes that the natural inducer, chloramphenicol, stalls a ribosome in the leader region of cat transcripts, which causes localized melting of the downstream stem-loop structure, allowing initiation of translation of the cat-86 coding sequence. Although it is established that ribosome stalling in the cat-86 leader can induce translation of the coding sequence, several subsequent steps predicted by the model remain to be experimentally confirmed. As a consequence, the present evidence for cat-86 regulation can also be explained by two other potential control devices, ribosome hopping and translational frameshifting. Here we describe experiments designed to determine whether the alternatives to translational attenuation regulate cat-86. The results obtained are inconsistent with both competing models and are consistent with predictions made by the translational attenuation model.
Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary-phase thermotolerance in Escherichia coli
Authors: Hengge-Aronis R , Klein W , Lange R , Rimmele M , Boos W
Published: November 2016

The rpoS (katF) gene of Escherichia coli encodes a putative sigma factor (sigma S) required for the expression of a variety of stationary phase-induced genes, for the development of stationary-phase stress resistance, and for long-term starvation survival (R. Lange and R. Hengge-Aronis, Mol. Microbiol. 5:49-59, 1991). Here we show that the genes otsA, otsB, treA, and osmB, previously known to be osmotically regulated, are also induced during transition into stationary phase in a sigma S-dependent manner. otsA and otsB, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively, are involved in sigma S-dependent stationary-phase thermotolerance. Neither sigma S nor trehalose, however, is required for the development of adaptive thermotolerance in growing cells, which might be controlled by sigma E.
Cell cycle parameters of Escherichia coli K-12
Authors: Allman R , Schjerven T , Boye E
Published: November 2016

A computer simulation routine was used to calculate the DNA distributions of exponentially growing cultures of Escherichia coli K-12. Simulated distributions were compared with distributions obtained experimentally by flow cytometry. Durations of the DNA replication period (C) and the postreplication period (D) were found by minimizing the difference between theoretical and experimental DNA histograms. It was demonstrated that the K-12 strains AB1157 and CM735 had C and D periods that differed widely from each other and from those of the previously measured strain B/rA, while strain MC1000 was shown to have the same durations of the C and D periods as strain B/rA. The variation between K-12 strains may explain the divergence in the literature regarding their C and D periods. Strains W3110 and AB1157 recA1 had DNA histograms that could not be adequately simulated by the classical Cooper-Helmstetter model, which is consistent with the asymmetrically located origin and terminus for W3110 and the asynchrony of initiation for AB1157 recA1.
Regulation and function of the Streptomyces plasmid pSN22 genes involved in pock formation and inviability
Authors: Kataoka M , Seki T , Yoshida T
Published: November 2016

pSN22 is an 11-kb multicopy plasmid from Streptomyces nigrifaciens which is being studied in Streptomyces lividans. A segment of about 7 kb of pSN22 contains five genes involved in conjugation. Three of them, traA, traB, and traR, are essential for plasmid transfer and for the mobilization of chromosomal markers (fertility), while the remaining two genes, spdA and spdB, merely enhance the efficiency of plasmid transfer, resulting in the formation of larger pocks. In vitro promoter-probing experiments identified a 550-bp BglII-SmaI DNA fragment with promoter activity in both orientations; Northern (RNA blot) hybridization identified corresponding divergent transcripts of 1 and 5.2 kb for traR and the traA-traB-spdB operon, respectively. The traR gene product repressed its own transcription and also the transcription of the traA-traB-spdB operon. Plasmids containing a functional traB gene could not "survive" without traR being present in the same cell either in cis or in trans, presumably because unregulated expression of traB is lethal to the host. Plasmids with a functional traA gene but without traR had a low transformation efficiency and inhibited the growth of host cells.
Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA
Authors: Rigling D , Van Alfen N K
Published: November 2016

Transmissible hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, is associated with cytoplasmic double-stranded-RNA (dsRNA) viruses. The fungal laccase has attracted interest because its activity is reduced in hypovirulent dsRNA-containing strains. A laccase cDNA clone was isolated by screening a cDNA expression library with antibodies against the purified extracellular laccase. The amino acid sequence deduced from part of the cDNA clone revealed high homology to other fungal laccases, especially to the Neurospora crassa laccase. A major laccase transcript 2.3 kb in length was detected in Northern (RNA) blots. In liquid culture, extracellular laccase activity was reduced by about 75% in the hypovirulent (dsRNA-free) strain EP155/2. In contrast, production of biomass was not affected by the dsRNA. Northern blot analysis indicated that dsRNA down regulates laccase biosynthesis by reducing laccase mRNA accumulation. The laccase gene is one of several developmentally regulated genes affected by the presence of dsRNA.
Regulation of the adhE gene, which encodes ethanol dehydrogenase in Escherichia coli
Authors: Chen Y M , Lin E C
Published: November 2016

The respiratory control of adhE, which encodes ethanol (alcohol) dehydrogenase in Escherichia coli, was examined at the transcriptional level by using various phi (adhE'-lacZ) adhE+ merodiploid strains. Expression of the adhE'-lacZ operon fusion was increased about eightfold by anaerobic growth. Under anaerobic growth conditions, provision of nitrate lowered the level of expression. Nitrate repression was more severe than aerobic repression. From analyses of various mutants with mutations related to nitrate reduction, nitrate repression appears to result from two effects. (i) When nitrate was present, NarL, the positive regulator of the nar operon, exerted a direct repression on adhE expression, which was demonstrable even aerobically. (ii) The chemical reduction of nitrate exerted an indirect effect by altering the cellular redox potential.
A novel L-serine deaminase activity in Escherichia coli K-12
Authors: Su H , Newman E B
Published: November 2016

We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely. Both mutations lie near 60.5 min on the E. coli genetic map. The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation.
Specific endonucleolytic cleavage of the mRNA for ribosomal protein S20 of Escherichia coli requires the product of the ams gene in vivo and in vitro
Authors: Mackie G A
Published: November 2016

Endonucleolytic cleavage is believed to initiate the degradation of most bacterial mRNAs, but with several exceptions, the enzymes responsible have yet to be identified. Crude (S-30) or partially fractionated extracts of Escherichia coli strains with reduced exonuclease activities catalyze the cleavage of a 372-residue RNA substrate containing the sequences coding for ribosomal protein S20 to yield a number of discrete products. The major product of 147 residues is obtained in 60 to 70% yield, is coterminal with the 3' end of the substrate, and is identical to an mRNA fragment previously characterized in vivo (G. A. Mackie, J. Bacteriol. 171:4112-4120, 1989). A number of other products of 150 to 340 residues are also formed, and the cleavage sites, typically N decreases AU sequences, have been identified in the S20 mRNA substrate by Northern (RNA) blotting and primer extension. All cleavages required a native rather than a denatured RNA substrate. The rate of cutting of the S20 mRNA substrate at the site yielding the prominent 147-residue product appears to be independent of cleavages at other sites. In addition, the activity of the putative endonuclease(s) depends strongly, both in vivo and in vitro, on the product of the ams gene, which is known to influence mRNA lifetimes in vivo. Taken together, the data show that the fractionated extract described here reproduces steps in the degradation of some mRNAs which occur in living cells.
Menaquinol-nitrate oxidoreductase of Bacillus halodenitrificans
Authors: Ketchum P A , Denariaz G , LeGall J , Payne W J
Published: November 2016

When grown anaerobically on nitrate-containing medium, Bacillus halodenitrificans exhibited a membrane-bound nitrate reductase (NR) that was solubilized by 2% Triton X-100 but not by 1% cholate or deoxycholate. Purification on columns of DE-52, hydroxylapatite, and Sephacryl S-300 yielded reduced methyl viologen NR (MVH-NR) with specific activities of 20 to 35 U/mg of protein that was stable when stored in 40% sucrose at -20 degrees C for 6 weeks. 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxypropone-1-sulfonat e (CHAPSO) and dodecyl-beta-D-maltoside stimulated enzyme activity three- to fourfold. Membrane extractions yielded purified NR that separated after electrophoresis into a 145-kDa alpha subunit, a 58-kDa beta subunit, and a 23-kDa gamma subunit. The electronic spectrum of dithionite-reduced, purified NR displayed peaks at 424.6, 527, and 557 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. Analyses revealed a molybdenum-heme-non-heme iron ratio of 1:1:8 for the NR and the presence of molybdopterin. Electron paramagnetic resonance (EPR) signals characteristic of iron-sulfur centers were detected at low temperature. EPR also revealed a minor signal centered in the g = 2 region of the spectra. Upon reduction with dithionite, the enzyme displayed signals at g = 2.064, 2.026, 1.906, and 1.888, indicative of the presence of low-potential iron-sulfur centers, which resolve most probably as two [4Fe-4S]+1 clusters. With menadiol as the substrate for nitrate reduction, the Km for nitrate was 50-fold less than that seen when MVH was the electron donor. The cytochrome b557-containing enzyme from B. halodenitrificans is characterized as a menaquinol-nitrate:oxidoreductase.
Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase
Authors: Dahl M K , Msadek T , Kunst F , Rapoport G
Published: November 2016

The DegS-DegU protein kinase-response regulator pair controls the expression of genes encoding degradative enzymes as well as other cellular functions in Bacillus subtilis. Both proteins were purified. The DegS protein was autophosphorylated and shown to transfer its phosphate to the DegU protein. Phosphoryl transfer to the wild-type DegU protein present in crude extracts was shown by adding 32P-labeled DegS to the reaction mixture. Under similar conditions, the modified proteins encoded by the degU24 and degU31 alleles presented a stronger phosphorylation signal compared with that of the wild-type DegU protein. This may suggest an increased phosphorylation of these modified proteins, responsible for the hyperproduction of degradative enzymes observed in the degU24 and degU31 mutants. However, the degU32 allele, which also leads to hyperproduction of degradative enzymes, encodes a modified DegU response regulator which seems not to be phosphorylatable. The expression of the hyperproduction phenotype of the degU32 mutant is still dependent on the presence of a functional DegS protein. DegS may therefore induce a conformational change of the degU32-encoded response regulator enabling this protein to stimulate degradative enzyme synthesis. Two alleles, degU122 and degU146, both leading to deficiency of degradative enzyme synthesis, seem to encode phosphorylatable and nonphosphorylatable DegU proteins, respectively.
Cell wall assembly in Bacillus megaterium: incorporation of new peptidoglycan by a monomer addition process
Authors: Gally D L , Hancock I C , Harwood C R , Archibald A R
Published: November 2016

The pattern of cross-linking in the peptidoglycan of Bacillus megaterium has been studied by the pulsed addition of radiolabeled diaminopimelic acid. The distribution of label in muropeptides, generated by digestion with Chalaropsis muramidase and separated by high-performance liquid chromatography, stabilized after 0.15 of a generation time. The proportion of label in the acceptor and donor positions of isolated muropeptide dimers stabilized over the same period of time. The results have led to the formulation a new model for the assembly of peptidoglycan into the cylindrical wall of B. megaterium by a monomer addition process. Single nascent glycan peptide strands form cross-linkages only with material at the inner surface of the wall. Maturation is a direct consequence of subsequent incorporation of further new glycan peptide strands, and there is no secondary cross-linking process. The initial distribution of muropeptides is constant. It follows that the final pattern of cross-linking in the wall is determined solely by, and can be forecast from, this repetitive pattern of incorporation. In a modified form, this model can also be applied to assembly of cell walls in rod-shaped gram-negative bacteria.
The RSF1 gene regulates septum formation in Saccharomyces cerevisiae
Authors: Veinot-Drebot L M , Johnston G C , Singer R A
Published: November 2016

Septum formation in the mitotic cell cycle of the budding yeast Saccharomyces cerevisiae occurs by conversion of the chitin ring, laid down at bud formation, into the primary septum. We show here that under certain conditions this septation is dependent on the newly identified RSF1 gene. However, cells harboring the rsf1-1 mutation accumulated in a postcytokinesis state, with delayed conversion of the chitin-rich annulus into the primary septum. This rsf1-1-mediated inhibition of septum formation only occurred under conditions of biosynthetic stress and was correlated with biosynthetically mediated inhibition of the cell-cycle regulatory step START. The RSF1 gene is distinct from the CHS2 chitin synthase gene that is responsible for septation, and thus RSF1 most likely encodes a regulator of chitin synthesis. We hypothesize that RSF1 activity facilitates septum formation during times of biosynthetic stress, to allow efficient septation even under these conditions.
Essential residues in the polar loop region of subunit c of Escherichia coli F1F0 ATP synthase defined by random oligonucleotide-primed mutagenesis
Authors: Fraga D , Fillingame R H
Published: November 2016

The conserved, polar loop region of subunit c of the Escherichia coli F1F0 ATP synthase is postulated to function in the coupling of proton translocation through F0 to ATP synthesis in F1. We have used a random mutagenesis procedure to define the essential residues in the region. Oligonucleotide-directed mutagenesis was carried out with a random mixture of mutant oligonucleotides, the oligonucleotide mixture being generated by chemical synthesis by using phosphoramidite nucleotide stocks that were contaminated with the other three nucleotides. Thirty mutant genes coding single-amino-acid substitutions in the region between Glu-37 and Leu-45 of subunit c were tested for function by analyzing the capacity of plasmids carrying the mutant genes to complement a Leu-4----amber subunit c mutant. All substitutions at the conserved Arg-41 residue resulted in loss of oxidative phosphorylation, i.e., transformants could not grow on a succinate carbon source. The other conserved residues were more tolerant to substitution, although most substitutions did result in impaired growth on succinate. We conclude that Arg-41 is essential in the function of the polar loop and that the ensemble of other conserved residues collectively maintain an optimal environment required for that function.
Nucleotide sequence and characterization of the sfs1 gene: sfs1 is involved in CRP*-dependent mal gene expression in Escherichia coli
Authors: Kawamukai M , Utsumi R , Takeda K , Higashi A , Matsuda H , Choi Y L , Komano T
Published: November 2016

We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfs1 through sfs12 (sugar fermentation stimulation). Previously, one (sfs7) of them was mapped at 65 min on the E. coli chromosome and identified as nlp, which has high homology to repressor protein (Ner) of Mu phage, which contains a putative DNA binding region (Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171:5222-5225, 1989). In this study, another gene (sfs1) located at 3.5 min was newly found and analyzed. The nucleotide sequence of sfs1 encoded a protein of 234 amino acids (molecular mass, 26,227 Da) which also has a putative DNA binding domain. Overexpression of the sfs1 gene in MK2001 resulted in a 10-fold increase of amylomaltase, which was still dependent on MalT. These results suggest that Sfs1 could be a new regulatory factor involved in maltose metabolism.
Acetate catabolism in the dissimilatory iron-reducing isolate GS-15
Authors: Champine J E , Goodwin S
Published: November 2016

Acetate-grown GS-15 whole-cell suspensions were disrupted with detergent and assayed for enzymes associated with acetate catabolism. Carbon monoxide dehydrogenase and formate dehydrogenase were not observed in GS-15. Catabolic levels of acetokinase and phosphotransacetylase were observed. Enzyme activities of the citric acid cycle, i.e., isocitrate dehydrogenase, 2-oxoglutarate sythase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were observed.
Nomenclature relating to restriction of modified DNA in Escherichia coli
Authors: Raleigh E A , Benner J , Bloom F , Braymer H D , DeCruz E , Dharmalingam K , Heitman J , Noyer Weidner M , Piekarowicz A , Kretz P L
Published: November 2016

At least three restriction systems that attack DNA containing naturally modified bases have been found in common Escherichia coli K-12 strains. These systems are McrA, McrBC, and Mrr. A brief summary of the genetic and phenotypic properties so far observed in laboratory strains is set forth, together with a proposed nomenclature for describing these properties.
A constitutive thiamine metabolism mutation, thi80, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae
Authors: Nishimura H , Kawasaki Y , Nosaka K , Kaneko Y , Iwashima A
Published: November 2016

We identified a strain carrying a recessive constitutive mutation (thi80-1) with an altered thiamine transport system, thiamine-repressible acid phosphatase, and several enzymes of thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole. The mutant shows markedly reduced activity of thiamine pyrophosphokinase (EC 2.7.6.2) and high resistance to oxythiamine, a thiamine antagonist whose potency depends on thiamine pyrophosphokinase activity. The intracellular thiamine pyrophosphate content of the mutant cells grown with exogenous thiamine (2 x 10(-7) M) was found to be about half that of the wild-type strain under the same conditions. These results suggest that the utilization and synthesis of thiamine in Saccharomyces cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level.
Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7
Authors: Benjamin R C , Voss J A , Kunz D A
Published: November 2016

Detailed restriction and nucleotide sequence analysis of the Pseudomonas putida TOL plasmid pDK1 xylE gene revealed significant homology with isofunctional xylE (81.5%) and nahH (78.0%) genes from the TOL pWW0 and NAH7 plasmids. The highest degrees of nucleotide and apparent amino acid conservation (82.2 and 86.4%, respectively) among all three genes were found to exist within a region comprising 264 nucleotides encoding the C terminus. A comparison of localized regions revealed significantly greater homology between xylEpWW0 and xylEpDK1 within the C-terminal region, whereas xylEpWW0 and nahH showed greater similarity at the N terminus. The possibility that xylEpWW0 may represent a genetic hybrid of xylEpDK1 and nahH is discussed.
Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli
Authors: Haney S A , Platko J V , Oxender D L , Calvo J M
Published: November 2016

We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.
ATP-dependent cadmium transport by the cadA cadmium resistance determinant in everted membrane vesicles of Bacillus subtilis
Authors: Tsai K J , Yoon K P , Lynn A R
Published: November 2016

Resistance to cadmium conferred by the staphylococcal plasmid pI258 occurs by means of energy-dependent efflux, resulting in decreased intracellular accumulation of cadmium. Recent sequence information suggested that efflux is mediated by a P-type ATPase. The cadA gene was previously expressed in Bacillus subtilis, conferring resistance to cadmium. Everted membrane vesicles were prepared from B. subtilis cells harboring either a plasmid containing the cadA system or the vector plasmid alone. 109Cd2+ transport into the everted membranes was measured in the presence of various energy sources. Cadmium transport was detected only in the presence of ATP as an energy source. The production of an electrochemical proton gradient (delta mu H+) by using NADH or phenazine methosulfate plus ascorbate was not able to drive transport. Reagents which dissipate delta pH abolished calcium transport due to the Ca2+/H+ antiporter but only partially inhibited cadmium transport. Inhibition of transport by the antibiotic bafilomycin A1 occurred at concentrations comparable to those which inhibit P-type ATPases. A band corresponding to the cadA gene product was identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibodies to the protein were prepared.
Role of glycosylation and deglycosylation in biosynthesis of and resistance to oleandomycin in the producer organism, Streptomyces antibioticus
Authors: Vilches C , Hernandez C , Mendez C , Salas J A
Published: November 2016

Cell extracts of Streptomyces antibioticus, an oleandomycin producer, can inactivate oleandomycin in the presence of UDP-glucose. The inactivation can be detected through the loss of biological activity or by alteration in the chromatographic mobility of the antibiotic. This enzyme activity also inactivates other macrolides (rosaramicin, methymycin, and lankamycin) which contain a free 2'-OH group in a monosaccharide linked to the lactone ring (with the exception of erythromycin), but not those which contain a disaccharide (tylosin, spiramycin, carbomycin, josamycin, niddamycin, and relomycin). Interestingly, the culture supernatant contains another enzyme activity capable of reactivating the glycosylated oleandomycin and regenerating the biological activity through the release of a glucose molecule. It is proposed that these two enzyme activities could be an integral part of the oleandomycin biosynthetic pathway.
Cloning, sequencing, expression, and functional studies of a 15,000-molecular-weight Haemophilus somnus antigen similar to Escherichia coli ribosomal protein S9
Authors: Theisen M , Potter A A
Published: November 2016

Haemophilus somnus is a gram-negative bacterium capable of causing a number of disease syndromes in cattle. This article describes the cloning and characterization of a gene coding for a 15,000-molecular-weight (15K) polypeptide which reacts strongly with antiserum against H. somnus. Analysis of plasmid-encoded polypeptides by polyacrylamide gel electrophoresis showed that the corresponding gene is the second in a transcriptional unit. The first gene codes for a protein with a molecular weight of approximately 17,000. Using antiserum against the two recombinant proteins, we could show that the natural proteins are predominantly present in purified ribosomes from H. somnus. The nucleotide sequence of both genes and flanking regions has been determined, and the deduced amino acid sequence of the two polypeptides was used to search for sequence homology in the GenBank data base. The 15K polypeptide showed 89% similarity to the Escherichia coli ribosomal protein S9, and the 17K polypeptide showed 94% similarity to the E. coli ribosomal protein L13. In E. coli, the corresponding genes constitute a bicistronic operon, with the same gene order as that found in H. somnus. A plasmid expressing the 15K protein was found to complement an E. coli rpsI mutation. When a frameshift mutation was introduced into the 15K protein gene, the resulting plasmid failed to complement this rpsI mutation, demonstrating functional homology between the 15K protein and S9 from E. coli. Downstream from the 15K protein gene is located another open reading frame, which could code for a polypeptide with a predicted molecular weight of 24,427. A protein with a similar molecular weight was detected in minicells containing the recombinant clone. This polypeptide is 69% similar to the stringent starvation protein (Ssp) of E. coli.
High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli
Authors: Dabert P , Ehrlich S D , Gruss A
Published: November 2016

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.
The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase
Authors: Funk C R , Zimniak L , Dowhan W
Published: November 2016

To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified.
Integration host factor is required for positive regulation of the tdc operon of Escherichia coli
Authors: Wu Y F , Datta P
Published: November 2016

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.
The CobII and CobIII regions of the cobalamin (vitamin B12) biosynthetic operon of Salmonella typhimurium
Authors: Escalante-Semerena J C , Johnson M G , Roth J R
Published: November 2016

A detailed deletion map of the CobII and CobIII regions of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2 has been constructed. The CobII region encodes functions needed for the synthesis of lower ligand 5,6-dimethylbenzimidazole (DMB); CobIII encodes functions needed for the synthesis of the nucleotide loop that joins DMB to the corrin macrocycle. The genetic analysis of 117 deletion, insertion, and point mutations indicates that (i) the CobII and CobIII mutations are contiguous--that is, they are grouped according to function; (ii) the CobII region is composed of four complementation groups (cobJKLM); (iii) cobM mutations do not complement mutations in any of the other three CobII groups; and (iv) CobIII mutations include three complementation groups that correspond to the cobU, cobS, and cobT genes.
Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts
Authors: Kawai S , Murao S , Mochizuki M , Shibuya I , Yano K , Takagi M
Published: November 2016

Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.
Separable domains define target cell specificities of an RTX hemolysin from Actinobacillus pleuropneumoniae
Authors: McWhinney D R , Chang Y F , Young R , Struck D K
Published: November 2016

The leukotoxin (LktA) from Pasteurella haemolytica and the hemolysin (AppA) from Actinobacillus pleuropneumoniae are members of a highly conserved family of cytolytic proteins produced by gram-negative bacteria. Despite the extensive homology between these gene products, LktA is specific for ruminant leukocytes while AppA, like other hemolysins, lyses erythrocytes and a variety of nucleated cells, including ruminant leukocytes. Both proteins require activation facilitated by the product of an accessory repeat toxin (RTX) C gene for optimal biological activity. We have constructed six genes encoding hybrid toxins by recombining domains of ltkA and appA and have examined the target cell specificities of the resulting hybrid proteins. Our results indicate that the leukocytic potential of AppA, like that of LktA, maps to the C-terminal half of the protein and is physically separable from the region specifying erythrocyte lysis. As a consequence, we were able to construct an RTX toxin capable of lysing erythrocytes but not leukocytes. The specificity of one hybrid was found to be dependent upon the RTX C gene used for activation. With appC activation, this hybrid toxin lysed both erythrocytes and leukocytes, while lktC activation produced a toxin which could attack only leukocytes. This is the first demonstration that the specificity of an RTX toxin can be determined by the process of C-mediated activation.
Transcriptionally active regions in the genome of the archaebacterium Haloferax volcanii
Authors: Trieselmann B A , Charlebois R L
Published: November 2016

Transcriptionally active regions of the Haloferax volcanii genome were mapped by hybridization of radiolabeled cDNA to Southern blots of our minimal set of overlapping cosmid clones covering 96% of the 4.1-Mbp genome. Transcription during exponential growth occurred in nearly every region of the 2,920-kbp chromosome. Large parts of the 690- and the 86-kbp plasmids were transcribed, but the 440-kbp plasmid showed little expression. Transcription after a 40-min heat shock at 65 degrees C was generally reduced, apart from a small set of strongly expressed loci all situated on the chromosome.
The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail
Authors: Heffernan E J , Harwood J , Fierer J , Guiney D
Published: November 2016

A fragment of the Salmonella typhimurium virulence plasmid containing the rck locus, when cloned in the recombinant cosmid pADE016, was shown previously to confer high-level complement resistance on both rough and smooth Escherichia coli, Salmonella minnesota, and S. typhimurium and was associated with the production of an outer membrane protein. We determined the nucleotide sequence of the fragment containing the rck locus. Mutations in the two major open reading frames confirmed that the complement resistance mediated by pADE016 was due to a single 555-bp rck gene encoding a 17-kDa outer membrane protein. Analysis of the rck gene revealed that the Rck outer membrane protein consisted of 185 amino acid residues, with a calculated postcleavage molecular mass of 17.4 kDa. Rck is homologous to a family of outer membrane proteins expressed in gram-negative bacteria, two of which have been associated with virulence-related phenotypes: PagC, required by S. typhimurium for survival in macrophages and for virulence in mice; and Ail, a product of the Yersinia enterocolitica chromosome capable of mediating bacterial adherence to and invasion of epithelial cell lines. Rck, most closely related to PagC, represents the third outer membrane protein in this five-member family with a distinct virulence-associated phenotype.
Cell surface modifications induced by calcium ion in the myxobacterium Stigmatella aurantiaca
Authors: Chang B Y , White D
Published: November 2016

Calcium ion induces in the myxobacterium Stigmatella aurantiaca the ability to glide on solid surfaces and to become cohesive (D. F. Gilmore and D. White, J. Bacteriol. 161:113-117, 1985; B. J. Womack, D. F. Gilmore, and D. White, J. Bacteriol. 171:6093-6096, 1989). The addition of calcium ion to the growth medium resulted in the formation of extracellular fibrils, the appearance in the membrane fractions of a 30-kDa protein, and the accumulation in a low-speed centrifugal pellet of 10 polypeptides that cross-reacted with affinity-purified antibody to one of the polypeptides. One of the polypeptides, a 55-kDa protein, was present in the membrane fraction of control cells not incubated with calcium ion and was apparently translocated to the extracellular matrix during incubation in medium containing calcium ion. The 55-kDa protein was immunologically related to a 65-kDa protein located on the fibrils of another myxobacterium, Myxococcus xanthus.
DNA gyrase, CS7.4, and the cold shock response in Escherichia coli
Authors: Jones P G , Krah R , Tafuri S R , Wolffe A P
Published: November 2016

We identify the A subunit of DNA gyrase as a cold shock protein whose synthesis is sustained following transfer of exponentially growing cultures of Escherichia coli from 37 to 10 degrees C. After a lag period in which its synthetic rate declines, synthesis of the A subunit of DNA gyrase increases relative to that of total protein. The duration of the lag period in synthesis and the synthetic rate of the A subunit appear dependent on the synthesis of a rapidly induced cold shock protein, CS7.4. The promoter of the gyrA gene contains specific binding sites for the CS7.4 protein, suggesting that CS7.4 acts at the transcriptional level to facilitate continued A-subunit synthesis. As synthesis of the B subunit of DNA gyrase is also sustained during cold shock, we suggest that an increase in the amount of DNA gyrase per cell might occur, which would potentially adapt the cells for growth at reduced temperatures (10 degrees C).
Transfer of Tn916 between Lactococcus lactis subsp. lactis strains is nontranspositional: evidence for a chromosomal fertility function in strain MG1363
Authors: Bringel F , Van Alstine G L , Scott J R
Published: November 2016

Lactococcus lactis subsp. lactis MG1363 can act as a conjugative donor of chromosomal markers. This requires a chromosomally located fertility function that we designate the lactococcal fertility factor (Laff). Using inter- and intrastrain crosses, we identified other L. lactis strains (LMO230 and MMS373) that appear to lack Laff. The selectable marker in our crosses was Tcr, carried by Tn916, a transposon present on the chromosome. The transfer of Tcr was not due to Tn916-encoded conjugative functions, because (i) L. lactis cannot act as a donor in Tn916-promoted conjugation (F. Bringel, G. L. Van Alstine, and J. R. Scott, Mol. Microbiol. 5:2983-2993, 1992) and (ii) transfer occurred when the Tcr marker was present in a Tn916 derivative containing a mutation, tra-641, that prevents Tn916-directed conjugation in any host. In addition, we isolated a strain in which Tn916 appears to be linked to Laff; this strain should be useful for further analysis of this fertility factor. In this strain, Tn916 is on the same 600-kb SmaI fragment as Clu, a fertility factor previously shown to promote lactose plasmid transfer in L. lactis. Thus, it is possible that Clu and Laff are identical.
Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus
Authors: Shao W , Wiegel J
Published: November 2016

A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75%. At 86 degrees C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.