bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli
Authors: Lin J , Lee I S , Frey J , Slonczewski J L , Foster J W
Published: November 2016

Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.
Characterization of the MetR binding sites for the glyA gene of Escherichia coli
Authors: Lorenz E , Stauffer G V
Published: November 2016

Sequence analysis of the glyA control region of Escherichia coli identified two regions with homology to the consensus binding sequence for MetR, a lysR family regulatory protein. Gel shift assays and DNase I protection assays verified that both sites bind MetR. Homocysteine, a coregulator for MetR, increased MetR binding to the glyA control region. The MetR binding sites were cloned into the pBend2 vector. Although the DNA did not show any significant intrinsic bend, MetR binding resulted in a bending angle of about 33 degrees. MetR-induced bending was independent of homocysteine. To verify that the MetR binding sites play a functional role in glyA expression, site-directed mutagenesis was used to alter the two binding sites in a lambda glyA-lacZ gene fusion phage. Changing the binding sites toward the consensus MetR binding sequence caused an increase in glyA-lacZ expression. Changing either binding site away from the consensus sequence caused a decrease in expression, suggesting that both sites are required for normal glyA regulation.
rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation
Authors: Condon C , Liveris D , Squires C , Schwartz I , Squires C L
Published: November 2016

Here we present evidence that only five of the seven rRNA operons present in Escherichia coli are necessary to support near-optimal growth on complex media. Seven rrn operons are necessary, however, for rapid adaptation to nutrient and temperature changes, suggesting it is the ability to adapt quickly to changing environmental conditions that has provided the selective pressure for the persistence of seven rrn operons in E. coli. We have also found that one consequence of rrn operon inactivation is a miscoordination of the concentrations of initiation factor IF3 and ribosomes.
Analysis of a Coxiella burnetti gene product that activates capsule synthesis in Escherichia coli: requirement for the heat shock chaperone DnaK and the two-component regulator RcsC
Authors: Zuber M , Hoover T A , Court D L
Published: November 2016

A 1.2-kb EcoRI genomic DNA fragment of Coxiella burnetti, when cloned onto a multicopy plasmid, was found to induce capsule synthesis (mucoidy) in Escherichia coli. Nucleotide sequence analysis revealed the presence of an open reading frame that could encode a protein of 270 amino acids. Insertion of a tet cassette into a unique NruI restriction site resulted in the loss of induction of mucoidy. Because of its ability to induce mucoidy, we designated this gene mucZ. Computer search for homologies to mucZ revealed 42% identity to an open reading frame located at 1 min of the E. coli chromosome. Interestingly, the C-terminal amino acid residues of MucZ share significant homology with the J domain of the DnaJ protein and its homologs, suggesting potential interactions between MucZ and components of the DnaK-chaperone machinery. Results presented in this paper suggest that E. coli requires DnaK-chaperone machinery for Lon-RcsA-mediated induction of capsule synthesis, as noticed first by S. Gottesman (personal communication). The induction caused by MucZ is independent of Lon-RcsA and is mediated through the two-component regulators RcsC and RcsB. DnaK and GrpE but not DnaJ are also required for the RcsB-mediated MucZ induction, and we propose that MucZ is a DnaJ-like chaperone protein that might be required for the formation of an active RcsA-RcsB complex and for the RcsC-dependent phosphorylation of RcsB. Discussions are presented that suggest three different roles for alternative forms of the DnaK-chaperone machinery in capsule production.
Suppression of the Fix- phenotype of Rhizobium meliloti exoB mutants by lpsZ is correlated to a modified expression of the K polysaccharide
Authors: Reuhs B L , Williams M N , Kim J S , Carlson R W , Côté F
Published: November 2016

The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception is Rhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix+) on alfalfa, an indeterminate nodule-forming plant. A mutation of lpsZ in AK631 results in a Fix- strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis. Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit. Rm5830 produced a polysaccharide with altered chromatographic and electrophoretic properties, indicating a difference in the molecular weight range. Similar results were obtained in a study of Rm1021, a wild-type isolate that lacks the lpsZ gene: the introduction of lpsZ into Rm1021 exoB (Rm6903) both suppresses the Fix- phenotype and results in a modified expression of the K polysaccharide. Chromatography and electrophoresis analysis showed that the polysaccharide extracted from Rm6903 lpsZ+ differed from that of Rm6903 in molecular weight range. Importantly, the effect of LpsZ is not structurally specific, as the introduction lpsZ+ into Rhizobium fredii USDA257 also resulted in a molecular weight range change in the structurally distinct K polysaccharide produced by that strain. This evidence suggests that LpsZ has a general effect on the size-specific expression of rhizobial K polysaccharides.
Contribution of gentamicin 2'-N-acetyltransferase to the O acetylation of peptidoglycan in Providencia stuartii
Authors: Payie K G , Rather P N , Clarke A J
Published: November 2016

A collection of Providencia stuartii mutants which either underexpress or overexpress aac(2')-Ia, the chromosomal gene coding for gentamicin 2'-N-acetyltransferase (EC 2.3.1.59), have been characterized phenotypically as possessing either lower or higher levels of peptidoglycan O acetylation, respectively, than the wild type. These mutants were subjected to both negative-staining and thin-section electron microscopy. P. stuartii PR100, with 42% O acetylation of peptidoglycan compared with 52% O acetylation in the wild type, appeared as irregular rods. In direct contrast, P. stuartii strains PR50.LM3 and PR51, with increased levels of peptidoglycan O acetylation (65 and 63%, respectively), appeared as coccobacilli and chain formers, respectively. Membrane blebbing was also observed with the chain-forming strain PR51. Thin sectioning of this mutant indicated that it was capable of proper constriction and separation. P. stuartii PM1, when grown to mid-exponential phase, did not have altered peptidoglycan O-acetylation levels, and cellular morphology remained similar to that of wild-type strains. However, continued growth into stationary phase resulted in a 15% increase in peptidoglycan O acetylation concomitant with a change of some cells from a rod-shaped to a coccobacillus-shaped morphology. The fact that these apparent morphological changes were directly related to levels of O acetylation support the view that this modification plays a role in the maintenance of peptidoglycan structure, presumably through the control of autolytic activity.
Substrate requirements for ErmC' methyltransferase activity
Authors: Zhong P , Pratt S D , Edalji R P , Walter K A , Holzman T F , Shivakumar A G , Katz L
Published: November 2016

ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.
Domain structure of phage P4 alpha protein deduced by mutational analysis
Authors: Ziegelin G , Linderoth N A , Calendar R , Lanka E
Published: November 2016

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.
Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon
Authors: Soncini F C , Véscovi E G , Groisman E A
Published: November 2016

The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus. beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited.
Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli
Authors: Yanofsky C , Horn V
Published: November 2016

The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be required by mutations at any one of three loci, two of which appear to be rho and rpoB.
Phosphate concentration regulates transcription of the Acinetobacter polyhydroxyalkanoic acid biosynthetic genes
Authors: Schembri M A , Bayly R C , Davies J K
Published: November 2016

The polyhydroxyalkanoic acid (PHA) biosynthetic gene locus was cloned and characterized from an Acinetobacter sp. isolated from activated sludge. Nucleotide sequence analysis identified three clustered genes, phaAAc (encoding a beta-ketothiolase), phaBAc (encoding an acetoacetyl coenzyme A reductase), and phaCAc (encoding a PHA synthase). In addition, an open reading frame (ORF1) with potential to encode a 13-kDa protein was identified within this locus. The sequence of the putative translational product of ORF1 does not show significant similarity to any sequences in the database. A plasmid containing the Acinetobacter pha locus conferred the ability to accumulate poly-beta-hydroxybutyrate on its Escherichia coli host. These genes appear to lie in an operon transcribed by two promoters upstream of phaBAc, an apparent constitutive promoter, and a second promoter induced by phosphate starvation and under pho regulon control. These as well as a number of additional potential transcription start points were identified by a combination of primer extension and promoter-chloramphenicol acetyltransferase gene fusion studies carried out in Acinetobacter or E. coli transformants.
Fine-structure deletion analysis of the transcriptional silencer of the proU operon of Salmonella typhimurium
Authors: Fletcher S A , Csonka L N
Published: November 2016

Transcriptional control of the osmotically regulated proU operon of Salmonella typhimurium is mediated in part by a transcriptional silencer downstream from the promoter (D.G. Overdier and L.N. Csonka, Proc. Natl. Acad. Sci. USA 89:3140-3144, 1992). We carried out a fine-structure deletion analysis to determine the structure and the position of the silencer, which demonstrated that this regulatory element is located between nucleotide positions +73 to +274 downstream from the transcription start site. The silencer appears to be made up of a number of components which have cumulative negative regulatory effects. Deletions or insertions of short nucleotide sequences (< 40 bp) between the proU promoter and the silencer do not disrupt repression exerted by the silencer, but long insertions (> or = 0.8 kbp) result in a high level of expression from the proU promoter, similar to that imparted by deletion of the entire silencer. The general DNA-binding protein H-NS is required for the full range of repression of the proU operon in media of low osmolality. Although in the presence of the silencer hns mutations increased basal expression from the proU promoter three- to sixfold, in the absence of the silencer they did not result in a substantial increase in basal expression from the proU promoter. Furthermore, deletion of the silencer in hns+ background was up to 10-fold more effective in increasing basal expression from the proU promoter than the hns mutations. These results indicate that osmotic control of the proU operon is dependent of some factor besides H-NS. We propose that the transcriptional regulation of this operon is effected in media of low osmolality by a protein which makes the promoter inaccessible to RNA polymerase by forming a complex containing the proU promoter and silencer.
Growth properties associated with A-U replacement of specific G-C base pairs in 16S rRNA from Escherichia coli
Authors: Triman K L
Published: November 2016

Mutations that disrupt each of seven specific G-C base pairs in 16S rRNA from Escherichia coli confer loss of expression of a plasmid-encoded 16S rRNA selectable marker (spectinomycin resistance). However, A-U replacement of G-C base pairs at nucleotides 359/52 or 1292/1245 in 16S rRNA permits normal expression of the marker. By contrast, A-U replacements at 146/176, 153/168, 350/339, or 1293/1244 are associated with loss of expression of the marker. These genetic studies are designed to determine the importance of specific base pairs by assessment of the structural and functional impairments of 16S rRNA molecules resulting from expression of base pair substitutions at these positions.
Interruption of the ferredoxin (flavodoxin) NADP+ oxidoreductase gene of Escherichia coli does not affect anaerobic growth but increases sensitivity to paraquat
Authors: Bianchi V , Haggård-Ljungquist E , Pontis E , Reichard P
Published: November 2016

Ferredoxin (flavodoxin) NADP+ oxidoreductase participates in methionine biosynthesis and in the function of two anaerobic enzymes, pyruvate formate-lyase and ribonucleotide reductase. We prepared insertion mutants of Escherichia coli lacking a functional enzyme. They do not require methionine and they grow well anaerobically, but they show increased sensitivity to paraquat.
Use of Tn5tac1 to clone a pel gene encoding a highly alkaline, asparagine-rich pectate lyase isozyme from an Erwinia chrysanthemi EC16 mutant with deletions affecting the major pectate lyase isozymes
Authors: Alfano J R , Ham J H , Collmer A
Published: November 2016

Erwinia chrysanthemi mutant CUCPB5047, delta(pelA pelE) delta(pelB pelC)::28bp delta(pelX) delta 4bp pehX::omega Cmr, was constructed, mutated with Tn5tac1, and screened for isopropyl-beta-D-thiogalactopyranoside-dependent pectate lyase (Pel) production. A Kmr SacI fragment from the hyperexpressing Pel+ mutant CUCPB5066 was cloned into Escherichia coli and sequenced. The gene identified, pelL, encodes a novel, asparagine-rich, highly alkaline enzyme that is similar in primary structure to PelX and in enzymological properties to PelE.
Cloning, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT
Authors: Gak E R , Chistoserdov A Y , Lidstrom M E
Published: November 2016

The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT.
Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain
Authors: Cecchini G , Sices H , Schröder I , Gunsalus R P
Published: November 2016

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.
Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability
Authors: Ahmer B M , Thomas M G , Larsen R A , Postle K
Published: November 2016

TonB protein appears to couple the electrochemical potential of the cytoplasmic membrane to active transport across the essentially unenergized outer membrane of gram-negative bacteria. ExbB protein has been identified as an auxiliary protein in this process. In this paper we show that ExbD protein, encoded by an adjacent gene in the exb cluster at 65', was also required for TonB-dependent energy transduction and, like ExbB, was required for the stability of TonB. The phenotypes of exbB exbD+ strains were essentially indistinguishable from the phenotypes of exbB+ exbD strains. Mutations in either gene resulted in the degradation of TonB protein and in decreased, but not entirely absent, sensitivities to colicins B and Ia and to bacteriophage phi 80. Evidence that the absence of ExbB or ExbD differentially affected the half-lives of newly synthesized and steady-state TonB was obtained. In the absence of ExbB or ExbD, newly synthesized TonB was degraded with a half-life of 5 to 10 min, while the half-life of TonB under steady-state conditions was significantly longer, approximately 30 min. These results were consistent with the idea that ExbB and ExbD play roles in the assembly of TonB into an energy-transducing complex. While interaction between TonB and ExbD was suggested by the effect of ExbD on TonB stability, interaction of ExbD with TonB was detected by neither in vivo cross-linking assays nor genetic tests for competition. Assays of a chromosomally encoded exbD::phoA fusion showed that exbB and exbD were transcribed as an operon, such that ExbD-PhoA levels in an exbB::Tn10 strain were reduced to 4% of the levels observed in an exbB+ strain under iron-limiting conditions. Residual ExbD-PhoA expression in an exbB::Tn10 strain was not iron regulated and may have originated from within the Tn10 element in exbB.
Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase
Authors: Ma K , Loessner H , Heider J , Johnson M K , Adams M W
Published: November 2016

The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91 degrees C by the fermentation of peptides and reduces elemental sulfur (S(o)) to H2S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S(o) in the medium, and no growth was observed in the absence of S(o). The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S(o) concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S(o) limitation. This was accompanied by increased amounts of H2 and alcohol (ethanol and butanol) from cultures grown with limiting S(o). Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M(r) of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under So limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.
Purification, characterization, and metabolic function of tungsten-containing aldehyde ferredoxin oxidoreductase from the hyperthermophilic and proteolytic archaeon Thermococcus strain ES-1
Authors: Heider J , Ma K , Adams M W
Published: November 2016

Thermococcus strain ES-1 is a strictly anaerobic, hyperthermophilic archaeon that grows at temperatures up to 91 degrees C by the fermentation of peptides. It is obligately dependent upon elemental sulfur (S(o)) for growth, which it reduces to H2S. Cell extracts contain high aldehyde oxidation activity with viologen dyes as electron acceptors. The enzyme responsible, which we term aldehyde ferredoxin oxidoreductase (AOR), has been purified to electrophoretic homogeneity. AOR is a homodimeric protein with a subunit M(r) of approximately 67,000. It contains molybdopterin and one W, four to five Fe, one Mg, and two P atoms per subunit. Electron paramagnetic resonance analyses of the reduced enzyme indicated the presence of a single [4Fe-4S]+ cluster with an S = 3/2 ground state. While AOR oxidized a wide range of aliphatic and aromatic aldehydes, those with the highest apparent kcat/Km values (> 10 microM-1S-1) were acetaldehyde, isovalerylaldehyde, and phenylacetaldehyde (Km values of < 100 microM). The apparent Km value for Thermococcus strain ES-1 ferredoxin was 10 microM (with crotonaldehyde as the substrate). Thermococcus strain ES-1 AOR also catalyzed the reduction of acetate (apparent Km of 1.8 mM) below pH 6.0 (with reduced methyl viologen as the electron donor) but at much less than 1% of the rate of the oxidative reaction (with benzyl viologen as the electron acceptor at pH 6.0 to 10.0). The properties of Thermococcus strain ES-1 AOR are very similar to those of AOR previously purified from the saccharolytic hyperthermophile Pyrococcus furiosus, in which AOR was proposed to oxidize glyceraldehyde as part of a novel glycolytic pathway (S. Mukund and M. W. W. Adams, J. Biol. Chem. 266:14208-14216, 1991). However, Thermococcus strain ES-1 is not known to metabolize carbohydrates, and glyceraldehyde was a very poor substrate (kcat/Km of < 0.2 microM-1S-1) for its AOR. The most efficient substrates for Thermococcus strain ES-1 AOR were the aldehyde derivatives of transaminated amino acids. This suggests that the enzyme functions to oxidize aldehydes generated during amino acid catabolism, although the possibility that AOR generates aldehydes from organic acids produced by fermentation cannot be ruled out.
Roles of bovine serum albumin and copper in the assay and stability of ammonia monooxygenase activity in vitro
Authors: Juliette L Y , Hyman M R , Arp D J
Published: November 2016

We investigated the effects of bovine serum albumin (BSA) on both the assay and the stability of ammonia-oxidizing activity in cell extracts of Nitrosomonas europaea. Ammonia-dependent O2 uptake activity of freshly prepared extracts did not require BSA. However, a dependence on BSA developed in extracts within a short time. The role of BSA in the assay of ammonia-oxidizing activity apparently is to absorb endogenous free fatty acids which are present in the extracts, because (i) only proteins which bind fatty acids, e.g., BSA or beta-lactoglobulin, supported ammonia-oxidizing activity; (ii) exogenous palmitoleic acid completely inhibited ammonia-dependent O2 uptake activity; (iii) the inhibition caused by palmitoleic acid was reversed only by proteins which bind fatty acids; and (iv) the concentration of endogenous free palmitoleic acid increased during aging of cell extracts. Additionally, the presence of BSA (10 mg/ml) or CuCl2 (500 microM) stabilized ammonia-dependent O2 uptake activity for 2 to 3 days at 4 degrees C. The stabilizing effect of BSA or CuCl2 was apparently due to an inhibition of lipolysis, because both additives inhibited the increase in concentrations of free palmitoleic acid in aging extracts. Other additives which are known to modify lipase activity were also found to stabilize ammonia-oxidizing activity. These additives included HgCl2, lecithin, and phenylmethylsulfonyl fluoride.
The C-terminal domain of NifL is sufficient to inhibit NifA activity
Authors: Narberhaus F , Lee H S , Schmitz R A , He L , Kustu S
Published: November 2016

In Klebsiella pneumoniae, transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself is regulated by the proteins NifA and NifL. NifA, an enhancer-binding protein, activates transcription by RNA polymerase containing the alternative sigma factor sigma 54. The central catalytic domain of NifA is sufficient for transcriptional activation, which can occur from solution. In vivo, NifL antagonizes the action of NifA in the presence of molecular oxygen or combined nitrogen. Inhibition has also been shown in vitro, but it was not responsive to environmental signals. Assuming a two-domain structure of NifL, we localized inhibition by NifL to its carboxy (C)-terminal domain, which is more soluble than the intact protein. The first line of evidence for this is that internal deletions of NifL containing an intact C-terminal domain were able to inhibit transcriptional activation by NifA in a coupled transcription-translation system. The second line of evidence is that the isolated C-terminal domain of NifL (assayed as a fusion to the soluble maltose-binding protein [MBP]) was sufficient to inhibit transcriptional activation by the central domain of NifA in a purified transcription system. The final line of evidence is that an MBP fusion to the C-terminal domain of NifL inhibited transcriptional activation by NifA in vivo. On the basis of these data, we postulate that the inhibitory function of NifL lies in its C-terminal domain and hence infer that this domain is responsible for interaction with NifA. Gel filtration experiments with MBP-NifL fusion derivatives lacking portions of the N- or C-terminal domain of the protein revealed that the C-terminal domain is the most soluble part of NifL. Up to 50% of two MBP-NifL truncations containing only the C-terminal domain appeared to be in a defined dimeric state.
Identity of the Escherichia coli cls and nov genes
Authors: Tropp B E , Ragolia L , Xia W , Dowhan W , Milkman R , Rudd K E , Ivanisević R , Savić D J
Published: November 2016

cls and nov mutants have similar increased sensitivities to novobiocin and reduced levels of cardiolipin, both of which can be corrected by plasmid-borne copies of either wild-type gene. A comparison of the DNA sequences of both genes further verifies their identity.
Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui
Authors: Fujino E , Fujino T , Karita S , Sakka K , Ohmiya K
Published: November 2016

The 6.2-kbp DNA fragment encoding the enzymes in the porphyrin synthesis pathway of a cellulolytic anaerobe, Clostridium josui, was cloned into Escherichia coli and sequenced. This fragment contained four hem genes, hemA, hemC, hemD, and hemB, in order, which were homologous to the corresponding genes from E. coli and Bacillus subtilis. A typical promoter sequence was found only upstream of hemA, suggesting that these four genes were under the control of this promoter as an operon. The hemA and hemD genes cloned from C. josui were able to complement the hemA and hemD mutations, respectively, of E. coli. The COOH-terminal region of C. josui HemA and the NH2-terminal region of C. josui HemD were homologous to E. coli CysG (Met-1 to Leu-151) and to E. coli CysG (Asp-213 to Phe-454) and Pseudomonas denitrificans CobA, respectively. Furthermore, the cloned 6.2-kbp DNA fragment complemented E. coli cysG mutants. These results suggested that both C. josui hemA and hemD encode bifunctional enzymes.
Expression of Shigella dysenteriae serotype 1 O-antigenic polysaccharide by Shigella flexneri aroD vaccine candidates and different S. flexneri serotypes
Authors: Fält I C , Schweda E K , Klee S , Singh M , Floderus E , Timmis K N , Lindberg A A
Published: November 2016

The potential utility of Shigella flexneri aroD vaccine candidates for the development of bi- or multivalent vaccines has been explored by the introduction of the genetic determinants rfp and rfb for heterologous O antigen polysaccharide from Shigella dysenteriae serotype 1. The serotype Y vaccine strain SFL124 expressed the heterologous antigen qualitatively and quantitatively well, qualitatively in the sense of the O antigen polysaccharide being correctly linked to the S. flexneri lipopolysaccharide R3 core oligosaccharide and quantitatively in the sense that typical yields were obtained, with ratios of homologous to heterologous O antigen being 4:1 for one construct and 1:1 for another. Moreover, both polysaccharide chains were shown to be linked to position O-4 of the subterminal D-glucose residue of the R3 core. In contrast to the hybrid serotype Y SFL124 derivatives, analogous derivatives of serotype 2a vaccine strain SFL1070 did not elaborate a complete heterologous O antigen. Such derivatives, and analogous derivatives of rough, O antigen-negative mutants of SFL1070, formed instead a hybrid lipopolysaccharide molecule consisting of the S. flexneri lipid A R3 core with a single repeat unit of the S. dysenteriae type 1 O antigen. Introduction of the determinants for the S. dysenteriae type 1 O antigen into a second serotype 2a strain and into strains representing other serotypes of S. flexneri, revealed the following for the expression of the heterologous O antigen: serotypes 1a, 1b, 2a, and 5a did not produce the heterologous O antigen, whereas serotypes 2b, 3a, 3b, 4a, 4b, 5b, and X did.
Unique cholesteryl glucosides in Helicobacter pylori: composition and structural analysis
Authors: Hirai Y , Haque M , Yoshida T , Yokota K , Yasuda T , Oguma K
Published: November 2016

A chloroform-methanol-extracted lipid of Helicobacter pylori was studied. Three kinds of glycolipids, accounting for about 25% (wt/wt) of the total lipid, were detected and identified to be cholesteryl glucosides. The structures of two of them were determined to be cholesteryl-alpha-D-glucopyranoside and cholesteryl-6-O-tetrade-canoyl-alpha-D-glucopyranoside, and the plausible structure of the third one was identified as cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside. Cholesteryl glucosides are very rare in animals and bacteria. Furthermore, those in H. pylori had an alpha-glycosidic linkage, which is rather unusual for natural glycosides, and a phosphate-linked cholesteryl glycoside like the cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside has not been reported previously. As the cholesterol glucosides were detected in strains obtained from diverse geographical locations, the presence of cholesteryl glucosides in H. pylori is a very unique and a characteristic feature of the species. These findings add a new facet to the physiology and biochemistry, especially the cholesterol and glucose metabolism, of H. pylori. Furthermore, the cholesteryl glucosides of H. pylori showed hemolytic activities.
Five promoters integrate control of the cob/pdu regulon in Salmonella typhimurium
Authors: Chen P , Ailion M , Bobik T , Stormo G , Roth J
Published: November 2016

Propanediol is degraded by a B12-dependent pathway in Salmonella typhimurium. The enzymes for this pathway are encoded in a small region (minute 41) that includes the pdu operon (controlling B12-dependent degradation of propanediol) and the divergent cob operon (controlling synthesis of cobalamin, B12). Expression of both operons is induced by propanediol and globally controlled by the ArcA and Crp systems. The region between the two operons encodes two proteins, PduF, a transporter of propanediol, and PocR, which mediates the induction of the regulon by propanediol. Insertion mutations between the pdu and cob operons have been characterized, and their exact positions have been correlated with mutant phenotypes. The region includes five promoters, four of which are controlled by the PocR protein and induced by propanediol. The cob and pdu operons each have one regulated promoter; the pduF gene is expressed from two regulated promoters (P1 and P2). The P1 and P2 transcripts extend beyond pduF to include the pocR gene; thus the PocR protein autoregulates its expression from these promoters. The fifth promoter, PPoc, is adjacent to the pocR gene and associated with a Crp binding site. We suggest that all global control of the regulon is exerted by regulating the level of PocR protein at the P1, P2, and PPoc promoters. A putative binding site for the PocR protein has been identified by computer analysis. Eight close matches to this proposed site were found in regions near the four promoters known to be regulated by PocR protein: PPdu, P1, P2, and PCob. A three-state model is proposed in which the regulon uses all five of its promoters to control expression.
Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes
Authors: van der Rest M E , de Vries Y , Poolman B , Konings W N
Published: November 2016

For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described. Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro. A genetically well-defined S. cerevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter. The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p. The levels of expression as well as the initial uptake rates were increased 20-fold compared with those in a yeast strain carrying only one chromosomal MAL locus. Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains. To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump. Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested.
Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of rhodanese
Authors: Kudlicki W , Odom O W , Merrill G , Kramer G , Hardesty B
Published: November 2016

A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation.
Comparative characterization of release factor RF-3 genes of Escherichia coli, Salmonella typhimurium, and Dichelobacter nodosus
Authors: Kawazu Y , Ito K , Matsumura K , Nakamura Y
Published: November 2016

The termination of protein synthesis in bacteria requires two codon-specific release factors, RF-1 and RF-2. A gene for a third factor, RF-3, that stimulates the RF-1 and RF-2 activities has been isolated from the gram-negative bacteria Escherichia coli and Dichelobacter nodosus. In this work, we isolated the RF-3 gene from Salmonella typhimurium and compared the three encoded RF-3 proteins by immunoblotting and intergeneric complementation and suppression. A murine polyclonal antibody against E. coli RF-3 reacted with both S. typhimurium and D. nodosus RF-3 proteins. The heterologous RF-3 genes complemented a null RF-3 mutation of E. coli regardless of having different sequence identities at the protein level. Additionally, multicopy expression of either of these RF-3 genes suppressed temperature-sensitive RF-2 mutations of E. coli and S. typhimurium by restoring adequate peptide chain release. These findings strongly suggest that the RF-3 proteins of these gram-negative bacteria share common structural and functional domains necessary for RF-3 activity and support the notion that RF-3 interacts functionally and/or physically with RF-2 during translation termination.
Molecular cloning, nucleotide sequence, and expression of a carboxypeptidase-encoding gene from the archaebacterium Sulfolobus solfataricus
Authors: Colombo S , Toietta G , Zecca L , Vanoni M , Tortora P
Published: November 2016

Mammalian metallocarboxypeptidases play key roles in major biological processes, such as digestive-protein degradation and specific proteolytic processing. A Sulfolobus solfataricus gene (cpsA) encoding a recently described zinc carboxypeptidase with an unusually broad substrate specificity was cloned, sequenced, and expressed in Escherichia coli. Despite the lack of overall sequence homology with known carboxypeptidases, seven homology blocks, including the Zn-coordinating and catalytic residues, were identified by multiple alignment with carboxypeptidases A, B, and T. S. solfataricus carboxypeptidase expressed in E. coli was found to be enzymatically active, and both its substrate specificity and thermostability were comparable to those of the purified S. solfataricus enzyme.
Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response
Authors: Fujimoto S , Tomita H , Wakamatsu E , Tanimoto K , Ike Y
Published: November 2016

The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Emr) or Tn916 (Tcr) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF- orfY-sea-1 with all but repB and traA oriented in the same (left-to-right) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10.
Mutations in GltC that increase Bacillus subtilis gltA expression
Authors: Belitsky B R , Sonenshein A L
Published: November 2016

Mutants with altered forms of GltC, a positive LysR-type regulator of Bacillus subtilis glutamate synthase gene expression, were isolated. The mutant GltC proteins stimulated expression from the wild-type gltA promoter region 1.5- to 2.0-fold and from mutant promoter regions up to 80-fold. Moreover, expression of gltA became much less dependent on a nitrogen source-associated signal.