bluuebunny/crossref_metadata_2025 huggingface dataset:
The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis
Authors: Mengin-Lecreulx D , Texier L , Rousseau M , van Heijenoort J
Published: November 2016

Physiological properties of the murG gene product of Escherichia coli were investigated. The inactivation of the murG gene rapidly inhibits peptidoglycan synthesis in exponentially growing cells. As a result, various alterations of cell shape are observed, and cell lysis finally occurs when the peptidoglycan content is 40% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors reveals the concomitant accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) and, to a lesser extent, that of undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide (lipid intermediate I), indicating that inhibition of peptidoglycan synthesis occurs after formation of the cytoplasmic precursors. The relative depletion of the second lipid intermediate, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)GlcNAc, shows that inactivation of the murG gene product does not prevent the formation of lipid intermediate I but inhibits the next reaction in which GlcNAc is transferred to lipid intermediate I. In vitro assays for phospho-MurNAc-pentapeptide translocase and N-acetylglucosaminyl transferase activities finally confirm the identification of the murG gene product as the transferase that catalyzes the conversion of lipid intermediate I to lipid intermediate II in the peptidoglycan synthesis pathway. Plasmids allowing for a high overproduction of the transferase and the determination of its N-terminal amino acid sequence were constructed. In cell fractionation experiments, the transferase is essentially associated with membranes when it is recovered.
RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli
Authors: Cannistraro V J , Kennell D
Published: November 2016

A previously unreported endoRNase present in the spheroplast fraction of Escherichia coli degraded homoribopolymers and small RNA oligonucleotides but not polymer RNA. Like the periplasmic endoRNase, RNase I, the enzyme cleaved the phosphodiester bond between any nucleotides; however, RNase I degraded polymer RNA as fast as homopolymers or oligomers. Both enzymes migrated as 27-kDa polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and could not be separated by various chromatographic procedures. In rna insertion mutants, both enzymes were completely missing; the spheroplast enzyme is called RNase I*, since it must be a form of RNase I. The two forms could be distinguished by physical treatments. RNase I could be activated by Zn2+, while RNase I* was inactive in the presence of Zn2+. RNase I was inactivated very slowly at 100 degrees C over a wide pH range, while RNase I* was inactivated slowly by heat at pH 4.0 but much more rapidly as the pH was increased to 8.0. In the presence of a thiol-binding agent, the inactivation at the higher pH values was much slower. These results suggest that RNase I*, but not RNase I, has free sulfhydryl groups. RNase I* activity in the cell against a common substrate was estimated to be several times that of RNase I. All four 2',3'-phosphomonoribonucleotides were identified in the soluble pools of growing cells. Such degradative products must arise from RNase I* activity. The activity would be suited for the terminal step in mRNA degradation, the elimination of the final oligonucleotide fragments, without jeopardizing the cell RNA. An enzyme with very similar specificity was found in Saccharomyces cerevisiae, suggesting that the activity may be widespread in nature.
Anaerobic regulation of transcription initiation in the arcDABC operon of Pseudomonas aeruginosa
Authors: Gamper M , Zimmermann A , Haas D
Published: November 2016

The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway, which is inducible under conditions of oxygen limitation and serves to generate ATP from arginine. The 5' end of arc mRNA extracted from anaerobically grown cells was determined by S1 and primer extension mapping. The transcription initiation site was located upstream of the arcD gene and 41.5 bp downstream of the center of the sequence TTGAC....ATCAG. This sequence, termed the ANR box, is similar to the consensus FNR recognition site of Escherichia coli. Transcription of the arc operon in P. aeruginosa was strongly decreased by a deletion of the TTGAC half site or by a mutation in the anr gene, which is known to code for the FNR-like regulatory protein ANR. During a transition from aerobic to anaerobic growth conditions, the concentrations of arc mRNAs and the levels of the ArcD and ArcA proteins rose in a parallel fashion. Mutational analysis of the arc promoter region led to the conclusion that the distance between the ANR box and the -10 promoter region is important for promoter strength, whereas the -35 region does not appear to be critical for arc promoter function. These findings and previous results indicate that anaerobic induction of the arc operon occurs at the level of transcription and requires the ANR box in cis and the ANR protein in trans.
Autoradiographic analysis of diaminopimelic acid incorporation in filamentous cells of Escherichia coli: repression of peptidoglycan synthesis around the nucleoid
Authors: Mulder E , Woldringh C L
Published: November 2016

Peptidoglycan synthesis rate in nonconstricting filaments of Escherichia coli dnaX(Ts) has been studied by autoradiography of incorporated [3H]diaminopimelic acid. Analysis of autoradiograms of whole cells and sacculi showed that peptidoglycan is synthesized at a reduced rate in the nucleoid-containing parts of these filaments. The lower rate of peptidoglycan synthesis in the cell center coincides with a higher local rate of protein synthesis. DNA-less cell formation in dnaX(Ts), dnaX(Ts) sfiA, and the minB minicell-forming mutant is accompanied by a local increase in peptidoglycan synthesis at the constriction site.
Purification and characterization of thin, aggregative fimbriae from Salmonella enteritidis
Authors: Collinson S K , Emödy L , Müller K H , Trust T J , Kay W W
Published: November 2016

Novel fimbriae were isolated and purified from the human enteropathogen Salmonella enteritidis 27655. These fimbriae were thin (measuring 3 to 4 nm in diameter), were extremely aggregative, and remained cell associated despite attempts to separate them from blended cells by centrifugation. The thin fimbriae were not solubilized in 5 M NaOH or in boiling 0.5% deoxycholate, 8 M urea, or 1 to 2% sodium dodecyl sulfate (SDS) with or without 5% beta-mercaptoethanol. Therefore, an unconventional purification procedure based on the removal of contaminating cell macromolecules in sonicated cell extracts by enzymatic digestion and preparative SDS-polyacrylamide gel electrophoresis (PAGE) was used. The insoluble fimbriae recovered from the well of the gel required depolymerization in formic acid prior to analysis by SDS-PAGE. Acid depolymerization revealed that the fimbriae were composed of fimbrin subunits, each with an apparent molecular mass of 17 kDa. Although their biochemical characteristics and amino acid composition were typical of fimbriae in general, these thin fimbriae were clearly distinct from other previously characterized fimbriae. Moreover, their fimbrin subunits had a unique N-terminal amino acid sequence. Native fimbriae on whole cells were specifically labeled with immune serum raised to the purified fimbriae. This immune serum also reacted with the denatured 17-kDa fimbrin protein in Western blots. The polyclonal immune serum did not cross-react with the other two native fimbrial types produced by this strain or with their respective fimbrins on Western blots (immunoblots). Therefore, these fimbriae represent the third fimbrial type produced by the enteropathogen S. enteritidis.
Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription
Authors: Wu L , Welker N E
Published: November 2016

We cloned and sequenced a fragment of the Bacillus stearothermophilus NUB36 chromosome that contains two open reading frames (ORFs) whose products were detected only in cells of cultures grown in complex medium at high temperature. The nucleotide sequence of the two ORFs exhibited significant identity to the sequence of the glnQ and glnH loci of the glutamine transport system in enteric bacteria. In addition, growth response to glutamine, sensitivity to the toxic glutamine analog gamma-L-glutamylhydrazide, and glutamine transport assays with parental strain NUB3621 and mutant strain NUB36500, in which the ORF1 coding segment in the chromosome was interrupted with the cat gene, demonstrated that glnQ and glnH encode proteins that are active in the glutamine transport system in B. stearothermophilus. The inferred promoter for the glnQH operon exhibited a low homology to the -35 and -10 regions of the consensus promoter sequences of Bacillus subtilis and Escherichia coli genes. In addition, the inferred promoter for the glnQH operon also exhibited a low homology with the consensus promoter sequence deduced from the sequences of the promoters of nine different genes from B. stearothermophilus. Transcription of the glnQH operon was activated in a nitrogen-rich medium at high temperature and inhibited under the same conditions at low temperature. Transcription of the glnQH operon was partially activated in a nitrogen-poor medium at low temperature. The region upstream from glnQ contains sequences that have a low homology with the nitrogen regulator I-binding sequences and the nitrogen-regulated promoters of enteric bacteria. The effect of temperature on the regulation of the glnQH operon is discussed.
Deamination of deoxycytidine nucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii
Authors: Speed R R , Winkler H H
Published: November 2016

Thymidylate biosynthesis via the methylation of dUMP is required for DNA replication in Rickettsia prowazekii, an obligate intracytoplasmic bacterium. In theory, dUMP synthesis could occur either by the deamination of deoxycytidine nucleotides or by the reduction of uridine nucleotides. Accordingly, the incorporation of both radiolabeled cytidine and uridine into the thymidylate of R. prowazekii was examined. After DNA hydrolysis and high-performance liquid chromatography, it was determined that 85% of the rickettsial thymidylate was derived from cytidine and the remaining 15% was derived from uridine. These findings were supported by the identification of a dCTP deaminase activity in extracts of R. prowazekii. Extracts of R. prowazekii deaminated 1.7 +/- 0.3 nmol of dCTP/min/mg of protein (a value calculated to suffice for rickettsial growth), and no measurable activity was observed with dCMP as the substrate.
Role of translation of the pheA leader peptide coding region in attenuation regulation of the Escherichia coli pheA gene
Authors: Gavini N , Pulakat L
Published: November 2016

In Escherichia coli, the expression of the pheA gene is regulated by attenuation of transcription. To study the molecular details of pheA attenuation, we introduced mutations in the pheA leader peptide coding region and analyzed their effects by using pheA promoter-lacZ gene transcription fusions (pheAp-lacZ). Mutations in the ribosome-binding site (pheAe1213) or in the translation initiation codon (pheAe24) of the pheA leader peptide coding region resulted in superattenuation of pheA expression. However, the presence of a stop codon upstream to the tandem phenylalanine codons (pheAe3334) led to an increase in the basal-level expression of pheA. This increase was further enhanced in the presence of prfA release factor mutant. The level of pheA expression in all three mutants was the same when cells were starved for phenylalanine. These results demonstrate that efficient translation of the pheA leader peptide coding region and the position of the ribosome on the leader transcript play decisive roles in the attenuation regulation of pheA.
Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase
Authors: Lauer G , Rudd E A , McKay D L , Ally A , Ally D , Backman K C
Published: November 2016

We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus. A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli. The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes. We have engineered the expression of the T. thermophilus gene in Escherichia coli, and we show that E. coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step.
Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli
Authors: Andrews A E , Dickson B , Lawley B , Cobbett C , Pittard A J
Published: November 2016

Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes. In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent. An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix. These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes. In the case of tyrP, moving the upstream box also affected activation. Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream. Practically no activation was seen at intermediate positions, such as +7 and -4. It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site.
Analysis of the host ranges of transposon bacteriophages Mu, MuhP1, and D108 by use of lipopolysaccharide mutants of Salmonella typhimurium LT2
Authors: Roncero C , Sanderson K E , Casadaban M J
Published: November 2016

The lipopolysaccharide receptors for the mutator bacteriophages Mu, MuhP1, and D108 were investigated with lipopolysaccharide mutants of Salmonella typhimurium LT2. Mu adsorbed only to mutants lacking the terminal O antigen but retaining the main chain sugars of the core; the side chain N-acetylglucosamine was not required. MuhP1 and D108 adsorbed partially to cells with the same receptors but adsorbed well only to cells with shorter lipopolysaccharides of the Rc and Rd1 chemotypes.
Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin
Authors: Béliveau C , Potvin C , Trudel J , Asselin A , Bellemare G
Published: November 2016

A Streptococcus faecalis genomic bank was obtained by partial digestion with MboI and cloning into the SalI restriction site of pTZ18R. Screening of about 60,000 Escherichia coli transformants for cell wall lysis activity was done by exposing recombinant colonies grown on medium containing lyophilized Micrococcus lysodeikticus cells to chloroform and toluene vapors in order to release proteins. Because this procedure provoked cell death, colonies could not be used directly for transformant recovery; however, recovery was achieved by partial purification of plasmid DNA from active colonies on the agar plate and transformation of E. coli competent cells. About 60 recombinants were found. One of them (pSH6500) codes for a lytic enzyme active against S. faecalis and M. lysodeikticus cell walls. A shorter clone (pSH4000) was obtained by deleting an EcoRI fragment from the 6.5-kb original insert, leaving a 4-kb EcoRI-MboI insert; this subclone expressed the same lytic activity. Sequencing of a portion of pSH4000 revealed a unique open reading frame of 2,013 nucleotides coding for a 641-amino-acid (74-kDa) polypeptide and containing four 204-nucleotide direct repeats.
Regulation of ompF porin expression by salicylate in Escherichia coli
Authors: Rosner J L , Chai T J , Foulds J
Published: November 2016

The expression of ompF, the gene encoding a major outer membrane protein of Escherichia coli, is regulated by various environmental factors. The mechanism by which salicylate (SAL) drastically reduces ompF expression was studied here by means of lacZ fusions to ompF, ompC, and micF, by sodium dodecyl sulfate-gel electrophoresis of outer membrane proteins, and by measurements of outer membrane permeability. Growth of E. coli in LB broth containing SAL strongly reduced ompF-specific translation of an ompF-lacZ fusion. The extent of this reduction varied with the SAL concentration from 64% at 0.5 mM to 95% at 2 mM and greater than 99% at 10 mM. ompF-lacZ transcription was not affected by SAL, whereas ompC-lacZ transcription was elevated by 70%. Since the micF transcript is antisense to a portion of the ompF transcript and is capable of decreasing the translation of ompF, the effect of SAL on micF transcription was measured in a micF-lacZ fusion strain. SAL-grown cells contained three- to fourfold more micF transcript during the logarithmic phase of growth than did the control cultures. However, micF was not absolutely required for the response to SAL. In micF-deleted strains, the effects of SAL on ompF translation, on OmpF in the outer membrane, and on outer membrane permeability were diminished but still evident. The effect of SAL on ompF expression was independent of the osmolarity of the medium and was epistatic to certain ompB regulatory mutations: the high levels of ompF expression found in envZ3 and ompR472 strains were greatly reduced by growth in SAL. Unexpectedly, the OmpC- phenotypes of these mutants were suppressed by SAL. Thus, growth in SAL severely decreases the translation of ompF while enhancing the transcription of micF and ompC. In this respect, SAL-grown cells resemble certain marA and tolC mutants that have high levels of micF and ompC transcripts and low levels of OmpF.
High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor
Authors: Butler C A , Gotschlich E C
Published: November 2016

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.
Integration host factor binds specifically to multiple sites in the ompB promoter of Escherichia coli and inhibits transcription
Authors: Tsui P , Huang L , Freundlich M
Published: November 2016

Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in gene regulation, site-specific recombination, and other processes in E. coli and some of its bacteriophages and plasmids. In the present study, we showed that IHF is a direct negative effector of the ompB operon of E. coli. Gel retardation experiments and DNase I footprinting studies revealed that IHF binds to three sites in the ompB promoter region. In vitro transcription from ompB promoter fragments was specifically blocked by IHF. In vivo experiments showed that IHF is a negative effector of ompB expression in growing cells. Analysis of IHF binding site mutations strongly suggested that IHF binding in the ompB promoter region is necessary for the negative effects seen in vivo.
Plasmids in diatom species
Authors: Hildebrand M , Corey D K , Ludwig J R , Kukel A , Feng T Y , Volcani B E
Published: November 2016

We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization previously characterized plasmids in Cylindrotheca fusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements.
Evolution of the ferric enterobactin receptor in gram-negative bacteria
Authors: Rutz J M , Abdullah T , Singh S P , Kalve V I , Klebba P E
Published: November 2016

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iron-deficient and replete cell envelopes, 59Fe-siderophore uptake studies, and Western immunoblots and cytofluorimetric analyses with monoclonal antibodies (MAbs), we surveyed a panel of gram-negative bacteria to identify outer membrane proteins that are structurally related to the Escherichia coli K-12 ferric enterobactin receptor, FepA. Antibodies within the panel identified FepA epitopes that are conserved among the majority of the bacteria tested, as well as epitopes present in only a few of the strains. In general, epitopes of FepA that are buried in the outer membrane bilayer were more conserved among gram-negative bacteria than epitopes that are exposed on the bacterial cell surface. The surface topology and tertiary structure of FepA are quite similar in E. coli and Shigella flexneri but differ in Salmonella typhimurium. Of the 18 different genera tested, 94% of the bacteria transported ferric enterobactin, including members of the previously unrecognized genera Citrobacter, Edwardsiella, Enterobacter, Haemophilus, Hafnia, Morganella, Neisseria, Proteus, Providencia, Serratia, and Yersinia. The ferric enterobactin receptor contains at least one buried epitope, recognized by MAb 2 (C. K. Murphy, V. I. Kalve, and P. E. Klebba, J. Bacteriol. 172:2736-2746, 1990), that is conserved within the structure of an iron-regulated cell envelope protein in all the bacteria that we have surveyed. With MAb 2, we identified and determined the Mr of cell envelope antigens that are immunologically related to E. coli FepA in all the gram-negative bacteria tested. Collectively, the library of anti-FepA MAbs showed unique patterns of reactivity with the different bacteria, allowing identification and discrimination of species within the following gram-negative genera: Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Haemophilus, Hafnia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia.
Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]
Authors: Dietrichs D , Meyer M , Rieth M , Andreesen J R
Published: November 2016

Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophilum and Clostridium litorale [corrected] was monitored by a new spectrophotometric assay. The procedure depended on a specific two- to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms. Protein PA isolated from E. acidaminophilum by 75Se labeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay. Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences. Depletion of thioredoxin from crude extracts of E. acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction. The former activity could be fully restored by adding thioredoxin. Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity. These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase.
Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway
Authors: Rosey E L , Oskouian B , Stewart G C
Published: November 2016

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus.
Molecular cloning and sequence of the thdF gene, which is involved in thiophene and furan oxidation by Escherichia coli
Authors: Alam K Y , Clark D P
Published: November 2016

Our previous work resulted in the isolation of mutant strains of Escherichia coli K-12 which were able to oxidize furans and thiophenes as a result of mutations in several novel genes. Some of the genes involved in thiophene oxidation were cloned into the multicopy vector pUC19. The plasmid pKA10 carries a 3.8-kb chromosomal fragment which encodes a previously undiscovered gene involved in thiophene oxidation. Three proteins with approximate molecular sizes of 48, 30, and 26 kDa were overproduced by cells carrying pKA10. Maxicell experiments and DNA sequence analysis indicated that the 48- and 26-kDa proteins are encoded by pKA10, whereas the 30-kDa protein is apparently chromosomally derived. A cassette specifying kanamycin resistance was inserted into various sites on pKA10. An insertion which abolished the 48-kDa protein also abolished thiophene oxidation. Chromosomal integration of pKA10::Kan allowed us to locate the chromosomal insert of pKA10 at 84 min on the E. coli genetic map by transduction. Since no previously identified genes involved in thiophene metabolism are located in this region, we designated the gene for the 48-kDa protein as thdF. Sequencing of the 3.8-kb insert revealed an overlap of several hundred bases with the regulatory and structural regions of the tnaA gene, which is also located at 84 min. The 26-kDa protein is probably truncated tnaA protein. An open reading frame corresponding to the 48-kDa thdF protein was located next to the tnaA gene, which encodes tryptophanase, but was transcribed in the opposite sense.
Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy
Authors: Poolman B , Molenaar D , Smid E J , Ubbink T , Abee T , Renault P P , Konings W N
Published: November 2016

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.
Regulation of phosphatidylglycerolphosphate synthase in Saccharomyces cerevisiae by factors affecting mitochondrial development
Authors: Gaynor P M , Hubbell S , Schmidt A J , Lina R A , Minskoff S A , Greenberg M L
Published: November 2016

Phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the first step in the synthesis of cardiolipin, an acidic phospholipid found in the mitochondrial inner membrane. In the yeast Saccharomyces cerevisiae, PGPS expression is coordinately regulated with general phospholipid synthesis and is repressed when cells are grown in the presence of the phospholipid precursor inositol (M. L. Greenberg, S. Hubbell, and C. Lam, Mol. Cell. Biol. 8:4773-4779, 1988). In this study, we examined the regulation of PGPS in growth conditions affecting mitochondrial development (carbon source, growth stage, and oxygen availability) and in strains with genetic lesions affecting mitochondrial function. PGPS derepressed two- to threefold when cells were grown in a nonfermentable carbon source (glycerol-ethanol), and this derepression was independent of the presence of inositol. PGPS derepressed two- to fourfold as cells entered the stationary phase of growth. Stationary-phase derepression occurred in both glucose- and glycerol-ethanol-grown cells and was slightly greater in cells grown in the presence of inositol and choline. PGPS expression in mitochondria was not affected when cells were grown in the absence of oxygen. In mutants lacking mitochondrial DNA [( rho0] mutants), PGPS activity was 30 to 70% less than in isogenic [rho+] strains. PGPS activity in [rho0] strains was subject to inositol-mediated repression. PGPS activity in [rho0] cell extracts was derepressed twofold as the [rho0] cells entered the stationary phase of growth. No growth phase derepression was observed in mitochondrial extracts of the [rho0] cells. Relative cardiolipin content increased in glycerol-ethanol-grown cells but was not affected by growth stage or by growth in the presence of the phospholipid precursors inositol and choline. These results demonstrate that (i) PGPS expression is regulated by factors affecting mitochondrial development; (ii) regulation of PGPS by these factors is independent of cross-pathway control; and (iii) PGPS expression is never fully repressed, even during anaerobic growth.
Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa
Authors: Tanaka E , Kawamoto S , Fukushima J , Hamajima K , Onishi H , Miyagi Y , Inami S , Morihara K , Okuda K
Published: November 2016

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.
Cloning, characterization, and expression in Escherichia coli of the genes encoding the cytochrome d oxidase complex from Azotobacter vinelandii
Authors: Moshiri F , Chawla A , Maier R J
Published: November 2016

Azotobacter vinelandii is a free-living nitrogen-fixing bacterium that has one of the highest respiratory rates of all aerobic organisms. Based on various physiological studies, a d-type cytochrome has been postulated to be the terminal oxidase of a vigorously respiring but apparently uncoupled branch of the electron transport system in the membranes of this organism. We cloned and characterized the structural genes of the two subunits of this oxidase. The deduced amino acid sequences of both subunits of the A. vinelandii oxidase have extensive regions of homology with those of the two subunits of the Escherichia coli cytochrome d complex. Most notably, the histidine residues proposed to be the axial ligands for the b hemes of the E. coli oxidase and an 11-amino-acid stretch proposed to be part of the ubiquinone binding site are all conserved in subunit I of the A. vinelandii oxidase. The A. vinelandii cytochrome d was expressed in a spectrally and functionally active form in the membranes of E. coli, under the control of the lac or tac promoter. The spectral features of the A. vinelandii cytochrome d expressed in E. coli are very similar to those of the E. coli cytochrome d. The expressed oxidase was active as a quinol oxidase and could reconstitute an NADH to oxygen electron transport chain.
Physiological effects of the fructose-1,6-diphosphate aldolase ts8 mutation on stable RNA synthesis in Escherichia coli
Authors: Singer M , Walter W A , Cali B M , Rouviere P , Liebke H H , Gourse R L , Gross C A
Published: November 2016

The conditional lethal mutations ts8 and h8 are located in fda, the gene encoding aldolase, and they inhibit RNA synthesis upon shift to the nonpermissive temperature. We demonstrate that both mutations preferentially inhibit stable RNA synthesis and that this inhibition occurs at the level of transcription initiation. The susceptibility of a promoter to the inhibitory effects of ts8 is correlated with the ability of the promoter to be growth rate regulated. This effect is independent of relA and spoT function. Inhibition is dependent upon glucose metabolism past the generation of glucose-6-phosphate; however, the mechanism of this effect is unknown.
A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans
Authors: Coque J J , Liras P , Laiz L , Martín J F
Published: November 2016

A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed.
Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases
Authors: Mock M , Crasnier M , Duflot E , Dumay V , Danchin A
Published: November 2016

The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.
Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport
Authors: Black P N
Published: November 2016

The fadL gene of Escherichia coli encodes an outer membrane protein (FadL) that plays a central role in the uptake of exogenous long-chain fatty acids. The nucleotide sequence of the fadL gene revealed a single open reading frame of 1,344 bp encoding a protein with 448 amino acid residues and a molecular weight of 48,831. The transcriptional start, analyzed by primer extension, was shown to be 95 bp upstream from the translational start. Apparent -10 and -35 regions were found at -12 and -37 bp upstream from the transcriptional start. Three regions with hyphenated dyad symmetry (two between the transcriptional start and the translational start and one upstream from the -10 and -35 regions) were identified that may play a role in the expression of fadL. The protein product of the fadL gene contained a signal sequence and signal peptidase I cleavage site similar to that defined for other E. coli outer membrane proteins. The N-terminal sequence of mature FadL protein was determined by automated amino acid sequencing of protein purified from the outer membrane of a strain harboring fadL under the control of a T7 RNA polymerase-responsive promoter. This amino acid sequence, Ala-Gly-Phe-Gln-Leu-Asn-Glu-Phe-Ser-Ser, verified the signal peptidase I cleavage site on pre-FadL and confirmed the N-terminal amino acid sequence of FadL predicted from the DNA sequence. Mature FadL contained 421 amino acid residues, giving a molecular weight of 45,969. The amino acid composition of FadL deduced from the DNA sequence suggested that this protein contained an abundance of hydrophobic amino acid residues and lacked cysteinyl residues. The hydrophobic amino acids within FadL were predicted to contribute to at least five regions of the protein with an overall hydrophobic character. The amino acid sequence of FadL was used to search GenBank for other proteins with amino acid sequence homology. These data demonstrated that FadL and the heat-modifiable outer membrane protein P1 of Haemophilus influenzae type b were 60.5% conserved and 42.0% identical over 438 amino acid residues.
Cloning and nucleic acid sequence of the Salmonella typhimurium pncB gene and structure of nicotinate phosphoribosyltransferase
Authors: Vinitsky A , Teng H , Grubmeyer C T
Published: November 2016

The pncB gene of Salmonella typhimurium, encoding nicotinate phosphoribosyltransferase (NAPRTase), was cloned on a 4.7-kb Sau3A fragment. The gene contains a 1,200-bp open reading frame coding for a 400-residue protein. Amino acid sequencing of the amino-terminal and two interior peptides of the purified protein confirmed the deduced sequence and revealed that the amino-terminal methionine residue was removed, giving a 399-residue mature protein of Mr 45,512. No signal sequence was observed in the predicted NAPRTase primary structure, suggesting that the enzyme is not periplasmic. The protein does not demonstrate clear sequence similarity to the other seven phosphoribosyltransferases of known primary structure and frustrates attempts to define a consensus 5-phosphoribosyl-1-pyrophosphate-binding region. The NAPRTase reaction is ATP stimulated, and the protein contains a carboxy-terminal sequence diagnostic of an ATP-binding site. An inverted repeat of the sequence TAAACAA observed in the proposed promoter region of pncB is also present in the promoter of nadA, which, like pncB, is also regulated by the NadR (NadI) repressor. The sequence may thus define an NadR repressor-binding site.
Mutations in DNA gyrase result in novobiocin resistance in halophilic archaebacteria
Authors: Holmes M L , Dyall-Smith M L
Published: November 2016

We have developed a cloning vector for use in halophilic archaebacteria which has a novobiocin resistance determinant as a selectable marker. The resistance determinant, which was derived from the genome of a resistant mutant strain, was mapped to a site within a 6.7-kb DNA clone by using a recombination assay and was sequenced. An open reading frame of 1.920 nucleotides (640 amino acids) was identified, with the predicted protein being highly homologous to the DNA gyrase B subunit (i.e., GyrB) of eubacteria. Three mutations were identified in the GyrB protein of the resistant mutant compared with the wild type (at amino acids 82, 122, and 137) which together enable Haloferax cells to grow in concentrations of novobiocin some 1,000 times higher than that possible for cells carrying only the wild-type enzyme. One base beyond the stop codon of gyrB was the start of gyrA, coding for the gyrase A subunit.
The genomes of the family Rhizobiaceae: size, stability, and rarely cutting restriction endonucleases
Authors: Sobral B W , Honeycutt R J , Atherly A G
Published: November 2016

The lack of high-resolution genetic or physical maps for the family Rhizobiaceae limits our understanding of this agronomically important bacterial family. On the basis of statistical analyses of DNA sequences of the Rhizobiaceae and direct evaluation by pulsed-field agarose gel electrophoresis (PFE), five restriction endonucleases with AT-rich target sites were identified as the most rarely cutting: AseI (5'-ATTAAT-3'), DraI (5'-TTTAAA-3'), SpeI (5'-ACTAGT-3'), SspI (5'-AATAAT-3'), and XbaI (5'-TCTAGA-3'). We computed the sizes of the genomes of Bradyrhizobium japonicum USDA 424 and Rhizobium meliloti 1021 by adding the sizes of DNA fragments generated by SpeI digests. The genome sizes of R. meliloti 1021 and B. japonicum USDA 424 were 5,379 +/- 282.5 kb and 6,195 +/- 192.4 kb, respectively. We also compared the organization of the genomes of free-living and bacteroid forms of B. japonicum. No differences between the PFE-resolved genomic fingerprints of free-living and mature (35 days after inoculation) bacteroids of B. japonicum USDA 123 and USDA 122 were observed. Also, B. japonicum USDA 123 genomic fingerprints were unchanged after passage through nodules and after maintenance on a rich growth medium for 100 generations. We conclude that large-scale DNA rearrangements are not seen in mature bacteroids or during free-living growth on rich growth media under laboratory conditions.