bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus
Authors: Aono S , Bryant F O , Adams M W
Published: November 2016

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.
Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro
Authors: Shavitt O , Livneh Z
Published: November 2016

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.
Overproduction of transposon Tn10-encoded tetracycline resistance protein results in cell death and loss of membrane potential
Authors: Eckert B , Beck C F
Published: November 2016

High-level expression of the Tn10 tetracycline resistance protein TetA in Escherichia coli caused partial collapse of the membrane potential, arrest of growth, and killing of the cells. Since alpha-methylglucoside transport was not affected, the overproduced TetA protein may cause not destruction of membrane structure but rather unrestricted translocation of protons and/or ions across the membrane.
Cloning and sequencing of the gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough
Authors: van Rooijen G J , Bruschi M , Voordouw G
Published: November 2016

The gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough was cloned by using two synthetic deoxyoligonucleotide probes. The amino acid sequence derived from the sequence of the gene differs from that reported by Bruschi and LeGall (Biochim. Biophys. Acta 271:48-60, 1972). Renewed protein sequencing confirmed the correctness of the DNA-derived sequence. The gene sequence indicates cytochrome c553 to be synthesized as a precursor protein with an NH2-terminal signal sequence of 24 residues.
Inhibition of protein synthesis transiently stimulates initiation of minichromosome replication in Escherichia coli
Authors: Weinberger M , Helmstetter C E
Published: November 2016

Replication of oriC-dependent minichromosomes was found to be transiently stimulated when protein synthesis was inhibited by the addition of chloramphenicol. Initiation of replication was also induced by amino acid starvation of relA mutant strains and a nutritional upshift. The results are explained on the basis that these treatments rendered RNA polymerase more available for participation in the initiation process. As a consequence, the oriC duplex may be transcriptionally activated to an open form, a necessary prerequisite for DNA polymerization.
Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene
Authors: Weickert M J , Chambliss G H
Published: November 2016

The amyR2 allele of the Bacillus subtilis alpha-amylase cis-regulatory region enhances production of amylase and transcription of amyE, the structural gene, by two- to threefold over amyR1. The amylase gene bearing each of these alleles was cloned on plasmids of about 10 to 15 copies per chromosome. Transcription of the cloned amylase gene by each amyR allele was activated at the end of exponential growth and was subject to catabolite repression by glucose. The amount of amylase produced was roughly proportional to the copy number of the plasmid, and cells containing the amyR2-bearing plasmid, pAR2, produced two- to threefold more amylase than cells with the amyR1 plasmid, pAMY10. Deletion of DNA 5' to the alpha-amylase promoter, including deletion of the A + T-rich inverted repeat found in amyR1 and amyR2, had no effect on expression or transcription of alpha-amylase. Deletion of DNA 3' to the amyR1 promoter did not impair temporal activation of chloramphenicol acetyltransferase in amyR1-cat-86 transcriptional fusions, but catabolite repression was abolished. When an 8-base-pair linker was inserted in pAMY10 at the same site from which the 3' deletion was made, amylase expression doubled and was repressed less by glucose. Both the deletion and the insertion disrupted four bases at the 3' end of the putative amylase operator region. Site-directed mutagenesis was used to change bases in the promoter-operator region of amyR1 to their amyR2 counterparts. Either change alone increased amylase production twofold, but only the change at +7, next to the linker insertion of 3' deletion site, yielded the increased amylase activity in the presence of glucose that is characteristic of the amyR2 strain. The double mutant behaved most like strains carrying the amyR2 allele.
Identification of subunits of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors
Authors: Klimpel K W , Lesley S A , Clark V L
Published: November 2016

Heparin-agarose and single-stranded DNA-cellulose chromatography were used to purify RNA polymerase 25-fold from Neisseria gonorrhoeae, and the activity of the polymerase was characterized in altered assay systems. The core subunits (beta, beta', and alpha) were tentatively identified as major proteins copurifying with polymerase activity. The identification of the core subunits was confirmed by Western (immunoblot) analysis with polyclonal antisera to Escherichia coli core RNA polymerase. Gonococcal sigma factor heterogeneity was examined by Western blot analysis with polyclonal antiserum to the major E. coli sigma factor, sigma 70, to the E. coli heat shock sigma factor, sigma 32, and with a monoclonal antiserum to Salmonella typhimurium NtrA (sigma 54). Purified RNA polymerase and whole-cell extracts from type 1, type 4, heat-shocked, and anaerobically grown gonococci were examined. Four putative gonococcal sigma factors were detected in purified RNA polymerase preparations and in whole-cell extracts from all cell types. Two of these bands appeared as a doublet, which had an estimated Mr of 80,000. A single lower-Mr band, estimated to be 40,000, was also present. All three of these bands reacted with antisera to E. coli sigma 70 and to E. coli sigma 32. A fourth gonococcal protein reacted solely with a highly specific monoclonal antibody to sigma 54 and had an Mr of 90,000. We conclude that N. gonorrhoeae may contain multiple sigma factors, which it may use to regulate gene expression.
Thermoregulation-dependent expression of Yersinia enterocolitica protein 1 imparts serum resistance to Escherichia coli K-12
Authors: Martinez R J
Published: November 2016

Resistance to the bactericidal action of normal human serum is one of the characteristics of virulent Yersinia enterocolitica. This property is attributable to the virulence plasmid harbored by pathogenic strains of the species. Serum resistance in Y. enterocolitica is thermoregulated, and its expression correlates well with the presence of virulence plasmid-encoded outer membrane proteins. To further examine the biochemical basis underlying resistance, we cloned a large segment (ca. 30 kilobases) of virulence plasmid DNA and studied the expression of plasmid-encoded outer membrane proteins in a serum-sensitive strain of Escherichia coli. The presence of the 160-kilodalton Y. enterocolitica-derived outer membrane protein 1 on E. coli transformants conferred a high degree of hydrophobicity, autoagglutinability, and resistance to serum killing. All of these properties were thermoregulated in E. coli with fidelity, suggesting that a functional thermoregulatory element was present in the cloned DNA. Elimination of protein 1 from the outer membrane of E. coli transformants by insertional inactivation of the structural gene with a Kanr gene cassette abrogated all of these properties and returned the serum-sensitive phenotype.
Normal growth and division of Escherichia coli with a reduced amount of murein
Authors: Prats R , de Pedro M A
Published: November 2016

The classical model of a murein monolayer for the structure of the murein sacculus of Escherichia coli is at present a controversial matter due to recent advances in the microscopic and biochemical knowledge of this cellular component (B. Glauner, J.-V. Höltje, and U. Schwarz, J. Biol. Chem. 263:10088-10095, 1988; M. Leduc, C. Frehel, E. Siegel, and J. van Heijenoort, manuscript in preparation). We have studied how the limitation in the supply of the murein-specific precursor meso-diaminopimelic acid (DAP) would affect cell growth and morphology as well as murein structure and content per unit of surface area in E. coli strains auxotrophic for DAP. Our observations indicated that the amount of murein per unit of cell surface area could be reduced by about 50% when the external concentration of DAP was equal to or lower than 0.5 microgram/ml. Cells with a reduced amount of murein did not show any detectable alteration in their morphology and characteristics of growth. Furthermore, no major alterations in murein structure were observed by high-pressure liquid chromatography analysis. However, the modification of murein that normally takes place at the initiation of the stationary phase was partially prevented. These results favor the alternative hypothesis of a thin, multilayered structure for the sacculus of E. coli rather than the monolayer model.
Identification of a second gene involved in global regulation of fumarate reductase and other nitrate-controlled genes for anaerobic respiration in Escherichia coli
Authors: Kalman L V , Gunsalus R P
Published: November 2016

Fumarate reductase catalyzes the final step of anaerobic electron transport in Escherichia coli when fumarate is used as a terminal electron acceptor. Transcription of the fumarate reductase operon (frdABCD) was repressed when cells were grown in the presence of either of the preferred terminal electron acceptors, oxygen or nitrate, and was stimulated modestly by fumarate. We have previously identified a locus called frdR which pleiotropically affects nitrate repression of fumarate reductase, trimethylamine N-oxide reductase, and alcohol dehydrogenase gene expression and nitrate induction of nitrate reductase expression (L. V. Kalman and R. P. Gunsalus, J. Bacteriol. 170:623-629, 1988). Transformation of various frdR mutants with plasmids identified two complementation groups, indicating that the frdR locus is composed of two genes. One class of mutants was not completely restored to wild-type frdA-lacZ expression or nitrate reductase induction when complemented with multicopy narX+ plasmids, whereas low-copy narX+ plasmid-containing strains were. A second class of frdR mutants was identified and shown to correspond to a previously described gene, narL (frdR2). Complementation of these strains with multicopy narL+ plasmids resulted in superrepression of frdA-lacZ expression and moderate elevation of nitrate reductase expression. Multicopy plasmids containing both narL+ and narX+ or only narL+ were able to complement narL mutants, whereas narX+ plasmids complemented narX mutants only when present in a copy number approximately equal to that of narL. Both narL and narX mutants retained normal oxygen control of frdA-lacZ expression. Both types of mutants are pleiotropic, as evidenced by derepressed levels of the fumarate reductase and trimethylamine N-oxide reductase enzymes and by defective induction of nitrate reductase when cells were grown in the presence of nitrate. These results indicate that both the narL and narX gene products must be present in a defined ratio in the cell. We conclude that these proteins interact to effect normal nitrate control of the anaerobic electron transport-associated operons. From these studies, we propose that narX encodes a nitrate sensor protein while narL encodes a DNA-binding regulatory protein which together function in a manner analogous to other two-component regulatory systems.
Identification of a umuDC locus in Salmonella typhimurium LT2
Authors: Smith C M , Eisenstadt E
Published: November 2016

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV light and many other DNA-damaging agents. The existence of a umuDC analog in Salmonella typhimurium has been questioned. With DNA probes to the E. coli umuD and umuC genes, we detected, by Southern blot hybridization, sequences similar to both of these genes in S. typhimurium LT2. We also confirmed that the presence of cloned E. coli umuD enhances the UV mutability and resistance of S. typhimurium. Our data strongly suggest that S. typhimurium contains a functional umuDC operon.
Differential expression of members of a cyanobacterial psbA gene family in response to light
Authors: Schaefer M R , Golden S S
Published: November 2016

The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains three psbA genes encoding two forms of the D1 protein: form I, the product of psbAI, differs from form II, the product of both psbAII and psbAIII, at 25 of 360 residues. D1 is essential for photosynthesis as a core component of the photosystem II reaction center. Translational gene fusions between each of the Synechococcus psbA genes and the Escherichia coli lacZ gene were inserted into the chromosome of wild-type Synechococcus sp. at the respective psbA loci to serve as in vivo reporters of psbA expression. beta-Galactosidase activities indicated differential expression of the psbA-lacZ gene fusions related to light availability. Expression of psbAI was 500-fold greater than expression of psbAII and 50-fold greater than psbAIII under similar conditions. As light intensity decreased from 600 microE.m-2.s-1 to 2 microE.m-2.s-1, expression of the psbAI reporter increased eightfold while expression of the psbAII and psbAIII reporters decreased 10-fold, suggesting differential production of the two forms of D1 in photosystem II in response to light availability. Relative levels of psbA-lacZ fusion transcripts directly reflected beta-galactosidase activities in the transformants, although the fusion transcripts were less stable than native psbA messages.
Autogenous regulation of the Escherichia coli ksgA gene at the level of translation
Authors: van Gemen B , Twisk J , van Knippenberg P H
Published: November 2016

Various plasmids that contain the Escherichia coli ksgA gene, which encodes a 16S rRNA adenosine dimethyltransferase (methylase), were constructed. In one of these plasmids, the DNA encoding the N-terminal part of the methylase was fused to the lacZ gene, and in another construct, the ksgA gene contained a deletion which resulted in a truncated version of the methylase. When a cell contained one plasmid directing the synthesis of the intact, active methylase and another plasmid encoding the methylase-beta-galactosidase protein, production of the latter product became strongly reduced. Likewise, synthesis of the truncated version of the methylase was diminished when the cell at the same time contained a plasmid producing the complete enzyme. These results were partly substantiated by in vitro experiments with a coupled transcription-translation assay system. By using a recently developed gel electrophoresis system for measuring protein-nucleic acid interactions, a specific binding of the ksgA methylase with its own mRNA could be established. Our results demonstrate that the expression of the ksgA gene can be, at least partly, autogenously controlled at the level of translation.
Symbiotic properties of rhizobia containing a flavonoid-independent hybrid nodD product
Authors: Spaink H P , Okker R J , Wijffelman C A , Tak T , Goosen-de Roo L , Pees E , van Brussel A A , Lugtenberg B J
Published: November 2016

A hybrid nodD gene consisting of 75% of the nodD1 gene of Rhizobium meliloti at the 5' end and 27% of the nodD gene of Rhizobium trifolii at the 3' end activates the six tested inducible nod promoters of Rhizobium leguminosarum, R. trifolii, or R. meliloti to maximal levels, even in the absence of flavonoids. In strains containing such a constitutive activating nodD gene, transcription of nod genes started at the same site as in flavonoid-induced strains containing a wild-type nodD gene. In contrast to heterologous wild-type nodD products, the constitutive activating nodD gene does not cause a limitation of the host range. Furthermore, R. leguminosarum, R. trifolii, and R. meliloti strains containing the constitutive activating nodD gene induce (pseudo) nodules on tropical leguminous plants. Comparison of the symbiotic properties of rhizobia containing the constitutive nodD hybrid gene with those of rhizobia containing various wild-type nodD genes indicates that the activation of the nodD product by flavonoids is of crucial importance during the process of infection thread formation and, surprisingly, also during nitrogen fixation.
Purification and properties of an endo-1,4-beta-glucanase from Clostridium josui
Authors: Fujino T , Sukhumavasi J , Sasaki T , Ohmiya K , Shimizu S
Published: November 2016

An enzyme active against carboxymethyl cellulose (CMC) was purified from the stationary-phase-culture supernatant of Clostridium josui grown in a medium containing ball-milled cellulose. The purification in the presence of 6 M urea yielded homogeneous enzyme after an approximately 50-fold increase in specific activity and a 13% yield. The enzyme had a molecular mass of 45 kilodaltons. The optimal temperature and pH of the enzyme against CMC were 60 degrees C and 6.8, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose to cellobiose and cellotriose but did not hydrolyze cellobiose or cellotriose. A microcrystalline cellulose, Avicel, was also hydrolyzed significantly, but the extent of hydrolysis was remarkably less than that of CMC. On the basis of these results, the enzyme purified here is one of the endo-1,4-beta-glucanases. The N-terminal amino acid sequence of the enzyme is Tyr-Asp-Ala-Ser-Leu-Lys-Pro-Asn-Leu-Gln-Ile-Pro-Gln-Lys-Asn-Ile-Pro-Asn- Asn-Asp-Ala-Val-Asn-Ile-Lys.
Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis
Authors: Mackenzie K F , Conway T , Aldrich H C , Ingram L O
Published: November 2016

The Zymomonas mobilis alcohol dehydrogenase II gene (adhB) was overexpressed 7- to 14-fold on a recombinant plasmid, accompanied by a small decrease in growth rate. A fragment containing the truncated gene with promoter reduced expression from the chromosomal gene as measured immunologically and enzymatically, consistent with the presence of a trans-active regulatory factor and positive regulatory control. Both the complete gene and the promoter fragment increased pyruvate decarboxylase and glucokinase activities, with no effect on alcohol dehydrogenase I or eight glycolytic enzymes. Tandem promoters from adhB expressed beta-galactosidase at higher levels than did either promoter alone in operon fusions. Addition of 50 microM zinc sulfate in minimal medium reduced the expression of adhB and of the operon fusions. Abundant but inactive alcohol dehydrogenase II was produced in iron-limited cells. This inactive enzyme did not form intracellular aggregates, and no morphological changes were apparent by transmission electron microscopy.
Role of Myxococcus xanthus cell surface antigen 1604 in development
Authors: Jarvis B W , Dworkin M
Published: November 2016

The inhibition of development of Myxococcus xanthus by monoclonal antibody (MAb) 1604 has been further investigated with two MAbs produced against the affinity-purified cell surface antigen (CSA) 1604. Both of these second-generation MAbs, 4070 and 4054, reacted with the same band at 150 kilodaltons (kDa) on Western immunoblots of lysed and reduced cells. This band was also identified by MAb 1604. However, the affinity-purified CSA was a complex of the two proteins (51 and 23 kDa) and lipopolysaccharide (LPS) that the 150-kDa material comprised. One of the three MAbs, 4070, reacted with LPS on Western immunoblots. Another MAb, 4054, reacted with the 23-kDa protein, and MAb 1604 reacted with the 51-kDa protein found in the CSA complex. Competitive binding studies verified that MAbs 4054 and 1604 identified different epitopes, and MAb 4070 probably reacted with a third epitope of the CSA 1604 complex. MAb 4054 blocked development, although not as thoroughly as MAb 1604 did, when added at 60 micrograms/ml to cells undergoing submerged development. In contrast, MAb 4070 prevented sporulation in submerged development and induced the cells to reaggregate in rings around the initial aggregation centers. A mutant strain of M. xanthus that is deficient in the epitope for MAb 1604 retained the epitope for MAb 4054. The affinity-purified antigen 1604, when added to cells at greater than or equal to 550 ng/ml, altered the appearance of the fruiting bodies and at higher concentrations prevented fruiting body formation. The CSA 1604 moiety responsible for this inhibitory effect is apparently a peptide constituent and not the LPS.
Posttranslational regulatory system for nitrogenase activity in Azospirillum spp
Authors: Fu H A , Hartmann A , Lowery R G , Fitzmaurice W P , Roberts G P , Burris R H
Published: November 2016

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.
Genetic and molecular analyses of the gene encoding staphylococcal enterotoxin D
Authors: Bayles K W , Iandolo J J
Published: November 2016

The gene (entD) encoding staphylococcal enterotoxin D (SED) has been located on a 27.6-kilobase penicillinase plasmid designated pIB485. This plasmid was present in all SED-producing strains tested. The entD gene was cloned on a 2.0-kilobase DNA fragment and was expressed in Escherichia coli. Sequence analysis of this fragment revealed an open reading frame that encoded a 258-amino-acid protein that possessed a 30-amino-acid signal peptide. The 228-amino-acid mature polypeptide had a molecular weight of 26,360 and contained a high degree of sequence similarity to the other staphylococcal enterotoxins. S1 nuclease mapping showed that transcription of entD was initiated 266 nucleotides upstream from the translation start codon. The entD gene was also shown to be activated by the staphylococcal regulatory element known as agr.
Temporal expression of transcription and relative copy number of plasmid pSFB-1 in Scytalidium flavo-brunneum
Authors: Caprioglio D R , Parks L W
Published: November 2016

Wild-type cultures of Scytalidium flavo-brunneum produce a 15-azasterol antifungal agent and a reddish brown pigment as secondary metabolites. Spontaneous mutants of S. flavo-brunneum that had simultaneously lost the ability to produce the 15-azasterol and the pigment were transformed with plasmid pSFB-1, which was obtained from wild-type S. flavo-brunneum. Each transformant possessed the plasmid and coincidentally reacquired azasterol and pigment production. Regulation of transcription and relative plasmid copy number was determined as a function of the culture cycle of the organism. Twenty-five-fold amplification of the plasmid was observed in the fungus during the stationary phase. RNA transcripts of 0.9, 1.0, 1.5, 2.0, and 2.5 kilobases were expressed from the plasmid by the organism. While differences in the temporal regulation of the transcripts were seen, all except the 1.0-kilobase transcript increased in abundance on entry of the culture into the stationary phase of growth.
Transcription of pfl is regulated by anaerobiosis, catabolite repression, pyruvate, and oxrA: pfl::Mu dA operon fusions of Salmonella typhimurium
Authors: Wong K K , Suen K L , Kwan H S
Published: November 2016

Pyruvate formate-lyase (EC 2.3.1.54), a key enzyme in the anaerobic metabolism of Salmonella typhimurium, catalyzes the conversion of pyruvate to acetyl coenzyme A and formate. pfl::Mu dA operon fusions were isolated for the study of transcriptional regulation. pfl was transcribed both aerobically and anaerobically, but the activity increased about sixfold under anaerobic conditions. The addition of pyruvate, formate, and acetate in nutrient broth did not have any effect on the anaerobic expression of pfl. However, the addition of pyruvate to minimal glucose medium increased the anaerobic expression of pfl. The expression of pfl varied in different growth media. Anaerobic expression of pfl was lower when the culture was grown in minimal glucose medium than when it was grown in nutrient broth. When Casamino Acids (Difco Laboratories, Detroit, Mich.) were added to minimal glucose medium, the expression of pfl increased proportionally with the amount of Casamino Acids added. The transcription of pfl was positively controlled by the oxrA gene product and was affected by both the cya and crp mutations. However, mutations in genes affecting the cyclic AMP-cyclic AMP receptor protein complex or oxrA could not completely abolish the anaerobic derepression of pfl. In merodiploid strains, pfl::Mu dA/F' pfl+, the beta-galactosidase activities were decreased. The mutations gyrA, oxrC, and oxrE, which affected anaerobic metabolism, did not affect anaerobic expression of pfl.
Transformation of a conditionally peptidoglycan-deficient mutant of Staphylococcus aureus with plasmid DNA
Authors: Nieuwlandt D T , Pattee P A
Published: November 2016

A simple and reliable method for polyethylene glycol-induced plasmid transformation of a temperature-sensitive peptidoglycan-deficient mutant of Staphylococcus aureus is described. The procedure uses strains carrying the tofA372 mutation grown under conditions that yield osmotically fragile cells capable of efficient wall regeneration. The peptidoglycan-deficient cells were transformed with plasmids pE194 and pI258 at frequencies comparable with those obtained with protoplasts prepared with lysostaphin treatment. A readily portable tofA372 mutation was constructed by isolating an insertion of the erythromycin resistance transposon Tn551 adjacent to tofA372. tofA372 was shown by protoplast fusion and transformation analyses to be in the gene order hly-421-omega [Chr::Tn551]1059-tofA372-uraB232-omega [Chr::Tn916]1101-thrB106 on the chromosome of S. aureus NCTC 8325.
Transcription initiation at multiple promoters of the pfl gene by E sigma 70-dependent transcription in vitro and heterologous expression in Pseudomonas putida in vivo
Authors: Sawers G , Wagner A F , Böck A
Published: November 2016

In vitro transcription experiments were used to provide further evidence that the gene encoding pyruvate formate-lyase (EC 2.3.1.54) from Escherichia coli is transcribed from seven promoters which cover a region of 1.2 kilobase pairs of DNA (G. Sawers and A. Böck, J. Bacteriol., 171:2485-2498, 1989). The results demonstrated that all promoters were recognized by the major RNA polymerase holoenzyme species E sigma 70 in vitro. Further corroboration for multiple functional promoters came from heterologous expression of the pfl operon in the obligate aerobe Pseudomonas putida. An immunological analysis indicated that the pyruvate formate-lyase protein was synthesized from a multicopy plasmid in P. putida, and S1 nuclease protection of RNA transcripts confirmed that all the pfl promoters on the plasmid were recognized by the host RNA polymerase. Transcription initiated at the same sites in P. putida and in E. coli for all the transcripts that were analyzed.
Heterologous expression of the alpha-amylase inhibitor gene cloned from an amplified genomic sequence of Streptomyces tendae
Authors: Koller K P , Riess G
Published: November 2016

The coding region for a secreted proteinaceous inhibitor of the human alpha-amylase (tendamistat; HOE 467) was identified by using a synthetic oligonucleotide probe. The gene is part of a 37-kilobase amplified genomic sequence found in an overproducing mutant of Streptomyces tendae. After subcloning, sequence analysis revealed an open reading frame of 312 base pairs preceded by a putative ribosome-binding site. The reading frame is 30 codons longer than necessary for the mature protein. This sequence coded for an amino-terminal extension of tendamistat and shows typical features of a signal peptide. After being cloned into Streptomyces vector plasmids and transformed to the heterologous host, Streptomyces lividans TK24, the gene was expressed, and the alpha-amylase inhibitor was correctly processed and secreted into the culture medium. The amount of secreted protein was dependent on the gene dosage and on the promoter arrangement.
Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine
Authors: Plamann M D , Stauffer G V
Published: November 2016

The methionine component of glyA gene regulation in Escherichia coli K-12 was investigated. The results indicate that the glyA gene is positively controlled by the metR gene product. Activation of glyA by the MetR protein requires homocysteine, an intermediate in methionine biosynthesis. The positive-acting metR regulatory system functions independently of a regulatory system shown previously to control glyA gene expression.
Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity
Authors: Somerville J E , Shatters R G , Kahn M L
Published: November 2016

The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia. Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species. These are encoded by the glnA and glnII genes, respectively. Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source. For two auxotrophs that lacked any detectable GS activity, R. meliloti DNA of the mutated region was cloned and partially characterized. Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity. One of the cloned regions was identified as glnII. An R. meliloti glnII mutant and an R. meliloti glnA glnII double mutant were constructed. Both formed effective nodules on alfalfa. This is unlike the B. japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop. However, the R. meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.
Degradation of phenol and m-toluate in Pseudomonas sp. strain EST1001 and its Pseudomonas putida transconjugants is determined by a multiplasmid system
Authors: Kivisaar M A , Habicht J K , Heinaru A L
Published: November 2016

The utilization of phenol, m-toluate, and salicylate (Phe+, mTol+, and Sal+ characters, respectively) in Pseudomonas sp. strain EST1001 is determined by the coordinated expression of genes placed in different plasmids, i.e., by a multiplasmid system. The natural multiplasmid strain EST1001 is phenotypically unstable. In its Phe-, mTol-, and Sal- segregants, the plasmid DNA underwent structural rearrangements without a marked loss of plasmid DNA, and the majority of segregants gave revertants. The genes specifying the degradation of phenol and m-toluate were transferable to P. putida PaW340, and in this strain a new multiplasmid system with definite structural changes was formed. The 17-kilobase transposable element, a part of the TOL plasmid pWWO present in the chromosome of PaW340, was inserted into the plasmid DNA in transconjugants. In addition, transconjugant EST1020 shared pWWO-like structures. Enzyme assays demonstrated that ortho-fission reactions were used by bacteria that grew on phenol, whereas m-toluate was catabolized by a meta-fission reaction. Salicylate was a functional inducer of the enzymes of both pathways. The expression of silent metabolic pathways of phenol or m-toluate degradation has been observed in EST1001 Phe- mTol+ and Phe+ mTol- transconjugants. The switchover of phenol degradation from the ortho- to the meta-pathway in EST1033 also showed the flexibility of genetic material in EST1001 transconjugants.
Evidence for unique DNA repair activity encoded by a cloned Serratia marcescens gene: suppression of Escherichia coli mutations that reduce repair of alkylated DNA
Authors: Murphy K E , Guzder S N , Braymer H D
Published: November 2016

A recombinant plasmid containing a Serratia marcescens DNA repair gene has been analyzed biochemically and genetically in Escherichia coli mutants deficient for repair of alkylated DNA. The cloned gene suppressed sensitivity to methyl methanesulfonate of an E. coli strain deficient in 3-methyladenine DNA glycosylases I and II (i.e., E. coli tag alkA) and two different E. coli recA mutants. Attempts to suppress the methyl methanesulfonate sensitivity of the E. coli recA mutant by using the cloned E. coli tag and alkA genes were not successful. Southern blot analysis did not reveal any homology between the S. marcescens gene and various known E. coli DNA repair genes. Biochemical analysis with the S. marcescens gene showed that the encoded DNA repair protein liberated 3-methyladenine from alkylated DNA, indicating that the DNA repair molecular is an S. marcescens 3-methyladenine DNA glycosylase. The ability to suppress both types of E. coli DNA repair mutations, however, suggests that the S. marcescens gene is a unique bacterial DNA repair gene.
Levels of DNA topoisomerases, single-stranded-DNA-binding protein, and DNA polymerase I in rho+ and rho-15 strains of Escherichia coli
Authors: Arnold G F , Phillips T A , Tessman I
Published: November 2016

The Escherichia coli rho-15 mutant, which is highly defective in transcription termination, was examined to see whether its reduced DNA superhelicity could be explained by altered expression of proteins that may affect DNA structure. Levels of DNA gyrase and topoisomerase I were normal; levels of single-stranded-DNA-binding protein, DNA polymerase I, and a protein tentatively identified as Lon were significantly altered.
Examination of enterotoxigenic Escherichia coli H10407 (colonization factor antigen I+) by scanning electron microscopy with conductive staining
Authors: Sherburne R , Armstrong G D
Published: November 2016

We have used the scanning electron microscope to examine enterotoxigenic Escherichia coli H10407, which expresses colonization factor antigen I pili. The use of low accelerating voltages and conductive staining procedures allowed us to obtain images of colonization factor antigen I pili and other structural details which were obscured by conventional gold-coating techniques.
Cloning and sequencing of an Escherichia coli gene, nlp, highly homologous to the ner genes of bacteriophages Mu and D108
Authors: Choi Y L , Nishida T , Kawamukai M , Utsumi R , Sakai H , Komano T
Published: November 2016

An nlp (Ner-like protein) gene was isolated from Escherichia coli. The nucleotide sequence of a 1,342-base-pair chromosomal DNA fragment containing the nlp gene was analyzed. It contained two open reading frames; one encoded 91 amino acid residues with an Mr of 10,361, and the other (ORFX) encoded 131 amino acid residues of the carboxyl-terminal region of a truncated polypeptide. The amino acid sequence deduced from the DNA sequence of nlp was highly homologous (62 to 63%) to the Ner proteins of bacteriophages Mu and D108. The amino-terminal region of Nlp deduced from the complete open reading frame contained a presumed DNA-binding region. The nlp gene was located at 69.3 min on the E. coli genetic map.
Cloning, nucleotide sequences, and identification of products of the Pseudomonas aeruginosa PAO bra genes, which encode the high-affinity branched-chain amino acid transport system
Authors: Hoshino T , Kose K
Published: November 2016

A DNA fragment of Pseudomonas aeruginosa PAO containing genes specifying the high-affinity branched-chain amino acid transport system (LIV-I) was isolated. The fragment contained the braC gene, encoding the binding protein for branched-chain amino acids, and the 4-kilobase DNA segment adjacent to 3' of braC. The nucleotide sequence of the 4-kilobase DNA fragment was determined and found to contain four open reading frames, designated braD, braE, braF, and braG. The braD and braE genes specify very hydrophobic proteins of 307 and 417 amino acid residues, respectively. The braD gene product showed extensive homology (67% identical) to the livH gene product, a component required for the Escherichia coli high-affinity branched-chain amino acid transport systems. The braF and braG genes encode proteins of 255 and 233 amino acids, respectively, both containing amino acid sequences typical of proteins with ATP-binding sites. By using a T7 RNA polymerase/promoter system together with plasmids having various deletions in the braDEFG region, the braD, braE, braF, and braG gene products were identified as proteins with apparent Mrs of 25,500, 34,000, 30,000, and 27,000, respectively. These proteins were found among cell membrane proteins on a sodium dodecyl sulfate-polyacrylamide gel stained with Coomassie blue.
Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA)
Authors: Bi E , Lutkenhaus J
Published: November 2016

In Escherichia coli, the ftsZ gene is thought to be an essential cell division gene. Several dominant mutations that make lon mutant cells refractory to the cell division inhibitor SulA, sulB9, sulB25, and sfiB114, have been mapped to the ftsZ gene. DNA sequence analysis of these mutations and the sfiB103 mutation confirmed that all of these mutations mapped within the ftsZ gene and revealed that the two sulB mutations were identical and by selection for resistance to higher levels of SulA, contained a second mutation within the ftsZ gene. We therefore propose that these mutations be redesignated ftsZ(Rsa) for resistance to SulA. A procedure involving mutagenesis of ftsZ cloned on low-copy-number vectors was used to isolate three additional ftsZ(Rsa) mutations. DNA sequence analysis of these mutations revealed that they were distinct from the previously isolated mutations. One of these mutations, ftsZ3(Rsa), led to an altered FtsZ protein that could no longer support cell growth but still conferred the Rsa phenotype in the presence of ftsZ+. In addition to being resistant to SulA, all ftsZ(Rsa) mutations also conferred resistance to a LacZ-FtsZ hybrid protein (ZZ). One possibility is that FtsZ functions as a multimer and that FtsZ(Rsa) mutant proteins have an increased ability for multimerization, making them resistant to SulA and ZZ.
Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction
Authors: Engel J N , Pollack J , Malik F , Ganem D
Published: November 2016

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.
Autoregulation of Escherichia coli purR requires two control sites downstream of the promoter
Authors: Rolfes R J , Zalkin H
Published: November 2016

The expression of Escherichia coli purR, which encodes the pur regulon repressor protein, is autoregulated. Autoregulation at the level of transcription requires two operator sites, designated purRo1 and purRo2 (O1 and O2). Operator O1 is in the region of DNA between the transcription start site and the site for translation initiation, and O2 is in the protein-coding region. The repressor protein binds noncooperatively to O1 with a sixfold-higher affinity than to O2, and saturation of O1 by the repressor precedes saturation of O2. Both O1 and O2 function in the two- to threefold autoregulation in vivo, as determined by measurement of beta-galactosidase and mRNA from purR-lacZ translational fusions. Of all the genes thus far known to be regulated by the Pur repressor, only purR employs a two-operator mechanism.
Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae
Authors: McCammon M T , Veenhuis M , Trapp S B , Goodman J M
Published: November 2016

Although peroxisomes are difficult to identify in Saccharomyces cerevisiae under ordinary growth conditions, they proliferate when cells are cultured on oleic acid. We used this finding to study the protein composition of these organelles in detail. Peroxisomes from oleic acid-grown cells were purified on a discontinuous sucrose gradient; they migrated to the 46 to 50% (wt/wt) sucrose interface. The peroxisomal fraction was identified morphologically and by the presence of all of the enzymes of the peroxisomal beta-oxidation pathway. These organelles also contained a significant but minor fraction of two enzymes of the glyoxylate pathway, malate synthase and malate dehydrogenase-2. The localization of malate synthase in peroxisomes was confirmed by immunoelectron microscopy. It is postulated that glyoxylate pathway enzymes are readily and preferentially released from peroxisomes upon cell lysis, accounting for their incomplete recovery from isolated organelles. Small uninduced peroxisomes from glycerol-grown cultures were detected on sucrose gradients by marker enzymes. Under these conditions, catalase, acyl-coenzyme A oxidase, and malate synthase cofractionated at equilibrium close to the mitochondrial peak, indicating smaller, less dense organelles than those from cells grown on oleic acid. Peroxisomal membranes from oleate cultures were purified by buoyant density centrifugation. Three abundant proteins of 24, 31, and 32 kilodaltons were observed.