bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel two-component transmembrane transcription control: regulation of iron dicitrate transport in Escherichia coli K-12
Authors: Van Hove B , Staudenmaier H , Braun V
Published: November 2016

Citrate and iron have to enter only the periplasmic space in order to induce the citrate-dependent iron(III) transport system of Escherichia coli. The five transport genes fecABCDE form an operon and are transcribed from fecA to fecE. Two genes, termed fecI and fecR, that mediate induction by iron(III) dicitrate have been identified upstream of fecA. The fecI gene encodes a protein of 173 amino acids (molecular weight, 19,478); the fecR gene encodes a protein of 317 amino acids (molecular weight, 35,529). Chromosomal fecI::Mu d1 mutants were unable to grow with iron(III) dicitrate as the sole iron source and synthesized no FecA outer membrane receptor protein. Growth was restored by transformation with plasmids encoding fecI or fecI and fecR. FecA and beta-galactosidase syntheses under transcription control of the fecB gene (fecB::Mu d1) were constitutive in fecI transformants and were regulated by iron(III) dicitrate in fecI fecR transformants. The amino acid sequence of the FecI protein contains a region close to the carboxy-terminal end for which a helix-turn-helix motif is predicted, which is typical for DNA-binding regulatory proteins. The FecI protein was found in the membrane, and the FecR protein was found in the periplasmic fraction. It is proposed that the FecR protein is the sensor that recognizes iron(III) dicitrate in the periplasm. The FecI protein activates fec gene expression by binding to the fec operator region. In the absence of citrate, FecR inactivates FecI. The lack of sequence homologies to other transmembrane signaling proteins and the location of the two proteins suggest a new type of transmembrane control mechanism.
Cloning and characterization of the aroA gene from Mycobacterium tuberculosis
Authors: Garbe T , Jones C , Charles I , Dougan G , Young D
Published: November 2016

The aroA gene from Mycobacterium tuberculosis has been cloned by complementation of an aroA mutant of Escherichia coli after lysogenization with a recombinant DNA library in the lambda gt11 vector. Detailed characterization of the M. tuberculosis aroA gene by nucleotide sequencing and by immunochemical analysis of the expressed product indicates that it encodes a 5-enolpyruvylshikimate-3-phosphate synthase that is structurally related to analogous enzymes from other bacterial, fungal, and plant sources. The potential use of the cloned gene in construction of genetically defined mutant strains of M. tuberculosis by gene replacement is proposed as a novel approach to the rational attenuation of mycobacterial pathogens and the possible development of new antimycobacterial vaccines.
In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600
Authors: Powlowski J , Shingler V
Published: November 2016

An in vitro study of the multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600 was performed. Phenol-stimulated oxygen uptake from crude extracts was strictly dependent on the addition of NAD(P)H and Fe2+ to assay mixtures. Five of six polypeptides required for growth on phenol were necessary for in vitro activity. One of the polypeptides was purified to homogeneity and found to be a flavin adenine dinucleotide containing iron-sulfur protein with significant sequence homology, at the amino terminus, to plant-type ferredoxins. This component, as in other oxygenase systems, probably functions to transfer electrons from NAD(P)H to the iron-requiring oxygenase component. Phenol hydroxylase from this organism is thus markedly different from bacterial flavoprotein monooxygenases commonly used for hydroxylation of other phenolic compounds, but bears a number of similarities to multicomponent oxygenase systems for unactivated compounds.
Identification of a heat shock promoter in the topA gene of Escherichia coli
Authors: Lesley S A , Jovanovich S B , Tse-Dinh Y C , Burgess R R
Published: November 2016

The transcriptional activity of the topA gene which codes for topoisomerase I was examined. An in vitro assay determined that the P1 promoter was dependent on the sigma 32 subunit of RNA polymerase. The transcriptional activity of the four topA promoters was examined by nuclease S1 mapping of the transcripts during a heat shock. This sigma 32-dependent promoter was shown to function as a heat shock promoter, although topoisomerase I is not a heat shock protein. A possible method of compensation of transcription activity by the other promoters to maintain the level of topoisomerase I during heat shock is proposed.
Enterobactin-mediated iron transport in Pseudomonas aeruginosa
Authors: Poole K , Young L , Neshat S
Published: November 2016

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence that modulation requires sequences downstream of the promoters of two vir-repressed genes of Bordetella pertussis
Authors: Beattie D T , Knapp S , Mekalanos J J
Published: November 2016

Gene expression in Bordetella pertussis is altered by environmental signals in a process called antigenic modulation. In the presence of modulating signals, expression of several known virulence factors and outer membrane proteins is coordinately reduced. From a bank of TnphoA fusions, we have identified five genes whose expression profiles are reciprocal of those of the major virulence determinants; that is, alkaline phosphatase activity is maximal during growth in the presence of the modulators nicotinic acid and MgSO4 (S. Knapp and J. J. Mekalanos, J. Bacteriol. 170:5059-5066, 1988). We have called these loci vir-repressed genes (vrg). Two of these gene fusions (vrg-6 and vrg-18) have been cloned in Escherichia coli, returned on low-copy-number plasmids to several strains of B. pertussis, and found to be regulated similarly to the fusions harbored on the chromosome. Deletions of the two vrg promoters were constructed and returned to B. pertussis. Regulation was maintained even when all but 24 nucleotides upstream of the vrg-18 initiation codon and 60 nucleotides upstream of the vrg-6 initiation codon were deleted, suggesting that cis-acting regulatory elements of these genes lie very near or within the coding region. We observed a 21-base palindromic sequence overlapping an 8-base direct repeat within the signal sequence coding region of vrg-6; insertion of a 6-bp linker in this region abolished regulation. These repetitive sequences are also at the site of greatest primary sequence identify between vrg-6 and vrg-18 and correspond to the signal sequence coding region. We propose models that involve recognition of this region by a vir-regulated gene product.
Characterization of cold-sensitive secY mutants of Escherichia coli
Authors: Baba T , Jacq A , Brickman E , Beckwith J , Taura T , Ueguchi C , Akiyama Y , Ito K
Published: November 2016

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.
Isolation and characterization of lipoteichoic acid, a cell envelope component involved in preventing phage adsorption, from Lactococcus lactis subsp. cremoris SK110
Authors: Sijtsma L , Wouters J T , Hellingwerf K J
Published: November 2016

The cell envelope of the phage-resistant Lactococcus lactis subsp. cremoris SK110 differed from its phage-sensitive variant by the presence of a galactosyl-containing component. This component was present in material obtained from SK110 by a mild alkali treatment. In a similar fraction extracted from SK112, no galactosyl-containing components were detected. With respect to gel permeation chromatography and electrophoretic mobility, identical characteristics of the alkali-extracted material and purified lipoteichoic acid (LTA) were measured. Chemical analysis of the latter component showed the absence of galactose in LTA isolated from SK112, whereas it was present in LTA obtained from SK110. In this paper, we propose that galactosyl-containing LTA is involved in preventing phage adsorption to L. lactis subsp. cremoris SK110.
Sequence of the pckA gene of Escherichia coli K-12: relevance to genetic and allosteric regulation and homology of E. coli phosphoenolpyruvate carboxykinase with the enzymes from Trypanosoma brucei and Saccharomyces cerevisiae
Authors: Medina V , Pontarollo R , Glaeske D , Tabel H , Goldie H
Published: November 2016

The sequence of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli K-12 and previous molecular weight determinations indicate that this allosteric enzyme is a monomer of Mr 51,316. The protein is homologous to ATP-dependent phosphoenolpyruvate carboxykinases from Trypanosoma brucei and Saccharomyces cerevisiae. A potential ATP binding site was conserved in all three sequences. A potential binding site for the allosteric activator, calcium, identified in the E. coli enzyme, was only partially conserved in T. brucei and S. cerevisiae, consistent with the observation that the enzymes from the latter organisms were not activated by calcium. The published sequence of the ompR and envZ genes from Salmonella typhimurium is followed by a partial sequence that is highly homologous to pckA from E. coli. The order of these genes and the direction of transcription of the presumptive S. typhimurium pckA gene are the same as those in E. coli. The potential calcium binding site of the E. coli enzyme is conserved in the partial predicted sequence of the S. typhimurium phosphoenolpyruvate carboxykinase, consistent with the observation that calcium activation of the S. typhimurium phosphoenolpyruvate carboxykinase is very similar to that observed for the E. coli enzyme. A pckA mRNA transcript was observed in stationary-phase cells but not in logarithmically growing cells. The mRNA start site was mapped relative to the sequence of the pckA structural gene.
Nucleotide sequence of the fruA gene, encoding the fructose permease of the Rhodobacter capsulatus phosphotransferase system, and analyses of the deduced protein sequence
Authors: Wu L F , Saier M H
Published: November 2016

The nucleotide sequence of the fruA gene, the terminal gene in the fructose operon of Rhodobacter capsulatus, is reported. This gene codes for the fructose permease (molecular weight, 58,575; 578 aminoacyl residues), the fructose enzyme II (IIFru) of the phosphoenolpyruvate-dependent phosphotransferase system. The deduced aminoacyl sequence of the encoded gene product was found to be 55% identical throughout most of its length with the fructose enzyme II of Escherichia coli, with some regions strongly conserved and others weakly conserved. Sequence comparisons revealed that the first 100 aminoacyl residues of both enzymes II were homologous to the second 100 residues, suggesting that an intragenic duplication of about 300 nucleotides had occurred during the evolution of IIFru prior to divergence of the E. coli and R. capsulatus genes. The protein contains only two cysteyl residues, and only one of these residues is conserved between the two proteins. This residue is therefore presumed to provide the active-site thiol group which may serve as the phosphorylation site. IIFru was found to exhibit regions of homology with sequenced enzymes II from other bacteria, including those specific for sucrose, beta-glucosides, mannitol, glucose, N-acetylglucosamine, and lactose. The degree of evolutionary divergence differed for different parts of the proteins, with certain transmembrane segments exhibiting high degrees of conservation. The hydrophobic domain of IIFru was also found to be similar to several uniport and antiport transporters of animals, including the human and mouse insulin-responsive glucose facilitators. These observations suggest that the mechanism of transmembrane transport may be similar for permeases catalyzing group translocation and facilitated diffusion.
Alveolysin, the thiol-activated toxin of Bacillus alvei, is homologous to listeriolysin O, perfringolysin O, pneumolysin, and streptolysin O and contains a single cysteine
Authors: Geoffroy C , Mengaud J , Alouf J E , Cossart P
Published: November 2016

The gene coding for alveolysin, the thiol-activated toxin produced by Bacillus alvei, has been cloned by means of an oligonucleotide based on the known N-terminal sequence of the secreted protein. The complete nucleotide sequence of the gene has been determined. The deduced amino acid sequence of alveolysin shows that alveolysin shares homologies with listeriolysin O, perfringolysin O, pneumolysin, and streptolysin O. Alveolysin, like the other members of the family, contains a single cysteine in the conserved peptide sequence ECTGLA WEWWR.
Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli
Authors: Wanner B L , Boline J A
Published: November 2016

The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively. Even though the E. coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E. coli K-12, as well as in several members of the E. coli reference (ECOR) collection which are closely related to K-12. For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source. Most E. coli strains, including E. coli B, are naturally Phn+; a few E. coli strains are Phn- and are deleted for phn DNA sequences. The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E. coli K-12 delta phn mutant. The phn(EcoB) DNA hybridized to overlapping lambda clones in the E. coli K-12 gene library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location. The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence. However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus.
The CAM-OCT plasmid enhances UV responses of Pseudomonas aeruginosa recA mutants
Authors: McBeth D L
Published: November 2016

The effect of the CAM-OCT plasmid on responses to UV irradiation of Pseudomonas aeruginosa recA mutants was characterized. Mutant alleles examined included rec-1, rec-2, and recA7::Tn501. The plasmid substantially enhanced both survival and mutagenesis of RecA- cells after treatment with UV light. Survival of the RecA-(CAM-OCT) cells after UV irradiation was intermediate between that seen in the wild-type P. aeruginosa PAO1 and the increased survival seen in PAO1(CAM-OCT) cells. Mutability was quantitated by the reversion to carbenicillin resistance of strains carrying a bla(Am) mutation on a derivative of plasmid RP1. UV-induced mutagenesis of CAM-OCT carrying recA mutants occurred at levels comparable to that seen in PAO1(CAM-OCT). The ability of CAM-OCT plasmid to suppress the recombination deficiency in recA mutants was tested by assaying for bacteriophage F116L-generalized transduction of a Tn7 insertion in the alkane utilization genes of CAM-OCT. Transduction of the Tn7 insertion was not detected in RecA-(CAM-OCT) strains but was easily seen in PAO1(CAM-OCT), indicating that the plasmid does not encode a recA analog. The results indicate that the CAM-OCT UV response genes are expressed in RecA- cells, which differs from results seen with other UV response-enhancing plasmids. The results suggest that CAM-OCT either encodes several UV responses genes itself or induces chromosomal UV response genes by an alternate mechanism.
Regulation of formate dehydrogenase activity in Methanococcus thermolithotrophicus
Authors: Sparling R , Daniels L
Published: November 2016

Methanococcus thermolithotrophicus can use either H2 or formate as the electron donor for methanogenesis from CO2. Resuspended-cell experiments revealed that the ability to use H2 as the source of electrons for methanogenesis was constitutive; cells grown on formate or H2-CO2 were equally capable of H2-CO2 methanogenesis. The ability to metabolize formate at high rates was observed only in cells previously grown on formate. Two such strains were distinguished: strain F and strain HF. Strain F was repeatedly grown exclusively on formate for over 3 years; this strain showed a constitutive capacity to metabolize formate to methane, even after subsequent repeated transfers to medium containing only H2-CO2. Strain HF could only metabolize formate to methane when grown in the presence of formate with no H2 present; this strain was recently derived from another strain (H) that had been exclusively grown on H2-CO2 and which upon initial transfer to formate medium could only metabolize formate to methane at a very slow rate. Initial adaptation of strain H to growth on formate was preceded by a long lag. The specific activities of hydrogenase and formate dehydrogenase in cell extracts derived from these different strains confirmed these findings. Similar levels of hydrogenase were observed in all strains, independent of the presence of H2 in the growth medium medium. High levels of formate dehydrogenase were also constitutive in strain F. Only low formate dehydrogenase activities were observed in strain H. High levels of formate dehydrogenase were observed in strain HF only when these cells were grown with formate in the absence of H2. In all strains the two- to threefold fluctuations of both hydrogenase and formate dehydrogenase cell-free activities were observed during growth, with peak activities reached in the middle of the exponential phase.
A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis
Authors: Breitling R , Dubnau D
Published: November 2016

In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein. The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3. The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain. The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells. The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter. Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene. A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium. This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase. In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.
Characterization of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae
Authors: Bowler C , Van Kaer L , Van Camp W , Van Montagu M , Inzé D , Dhaese P
Published: November 2016

Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could function in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.
Identification and characterization of gyrB mutants of Escherichia coli that are defective in partitioning of mini-F plasmids
Authors: Ogura T , Niki H , Mori H , Morita M , Hasegawa M , Ichinose C , Hiraga S
Published: November 2016

hopA mutants, which have been suggested to be defective in mini-F plasmid partitioning (H. Niki, C. Ichinose, T. Ogura, H. Mori, M. Morita, M. Hasegawa, N. Kusukawa, and S. Hiraga, J. Bacteriol. 170:5272-5278, 1988), were found to carry mutations in the gyrB gene, coding for the B subunit of DNA gyrase. In gyrB(HopA) mutants, relaxation of the superhelicity of plasmids, increased IncG incompatibility, and increased SopB protein production were observed. It is suggested that altered expression of the sop genes, which is due to relaxation of the mini-F plasmid DNA, causes both defective partitioning of the mini-F plasmids and increased IncG incompatibility in gyrB(HopA) mutants.
In vivo expression of the Pseudomonas stutzeri maltotetraose-forming amylase gene (amyP)
Authors: Fujita M , Futai M , Amemura A
Published: November 2016

Northern hybridization and S1 nuclease mapping revealed that the amyP gene coding for maltotetraose-forming amylase of Pseudomonas stutzeri MO-19 is transcribed as a monocistronic mRNA of 2.0 kilobases and that the transcription start site is located 81 base pairs upstream from the first nucleotide of the initiation codon. The amyP gene was expressed weakly in Escherichia coli, and transcription started 49 base pairs downstream of the P. stutzeri MO-19 transcription start site. Synthesis of the amylase in P. stutzeri MO-19 was induced by the addition of maltose to the culture medium and was repressed by the addition of glucose. The induction by maltose was shown to be result of transcription induction of the amyP gene. In contrast, glucose did not repress transcription initiation of amyP, indicating that it controls synthesis of the enzyme, probably at the posttranscriptional level.
High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria
Authors: Schäfer A , Kalinowski J , Simon R , Seep-Feldhaus A H , Pühler A
Published: November 2016

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.
A stable variant of Simonsiella muelleri with unusual colonial and cellular morphology
Authors: Whitehouse R L , Merrill H , Jackson M C , Jackson H
Published: November 2016

The unusual morphology and cellular arrangement of a member of the genus Simonsiella is described. The organism is characterized by the formation of very long trichomes, which can be greater than 1,000 microns in length.
Recombination of Salmonella phase 1 flagellin genes generates new serovars
Authors: Smith N H , Beltran P , Selander R K
Published: November 2016

To determine the evolutionary mechanisms generating serotypic diversity in Salmonella strains, we sequenced the central, antigen-determining part of the phase 1 flagellin gene (fliC) in strains of several serovars for which estimates of chromosomal genomic relatedness had been obtained by multilocus enzyme electrophoresis. The nucleotide sequence of this region was identical in several chromosomally divergent strains of Salmonella heidelberg (phase 1 antigen r) but differed by 19% from the corresponding and similarly invariant sequence in strains of the closely related serovar Salmonella typhimurium (phase 1 antigen i). Mutational drift of the sequence present in the common ancestor is unlikely to have generated the difference between the phase 1 flagellins of these two serovars, which we attribute instead to a recombination event. This interpretation is supported by evidence that Salmonella strains of very diverse chromosomal backgrounds but similar phase 1 antigens may have closely similar nucleotide sequences for this highly polymorphic region. We suggest that lateral transfer and recombination of phase 1 flagellin genes is a major evolutionary mechanism generating new Salmonella serovars.
Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus
Authors: Gassner G T , Dickie J P , Hamerski D A , Magnuson J K , Anderson J S
Published: November 2016

Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB3H4 followed by complete acid hydrolysis yielded [3H] glucosaminitol from the true reducing end residue of teichuronic acid and [3H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.
Aerobic regulation of the Escherichia coli tonB gene by changes in iron availability and the fur locus
Authors: Postle K
Published: November 2016

The tonB gene is required for the transport of several different iron-siderophore complexes across the Escherichia coli outer membrane. In this study, transcriptional regulation of the tonB gene was investigated by using three different tonB-lacZ fusions to monitor tonB expression under aerobic conditions and in the presence of a wild-type tonB gene. Prior work by other laboratories suggests that tonB is expressed at low constitutive levels regardless of changes in iron availability or the fur locus. In contrast, these data show that tonB transcription is repressed threefold by growth in the presence of FeCl3 compared with growth in the presence of the iron chelator dipyridyl and that this repression requires the fur locus. A 168-base-pair DNA fragment carrying the tonB promoter was sufficient for the observed transcriptional regulation. In addition, the tonB gene appeared to have a substantially stronger promoter than previously recognized. The inability of other laboratories to detect tonB transcription regulation appears to be due to the extremely slow growth of iron-starved tonB strains and the use of Mu d1(lac Apr)- or lambda plac Mu53-generated fusions that encode a thermolabile TrpA-LacZ hybrid protein. The data also suggest that the previously reported growth phase regulation of tonB occurs only in media with intermediate levels of available iron and is due to iron starvation-induced derepression as the culture approaches stationary phase.
Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus
Authors: Weinberg R A , Zusman D R
Published: November 2016

One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and germination.
Interdomain hybrid Tet proteins confer tetracycline resistance only when they are derived from closely related members of the tet gene family
Authors: Rubin R A , Levy S B
Published: November 2016

Inner membrane Tet proteins encoded by tet genes in gram-negative bacteria mediate resistance to tetracycline (Tcr) by directing its export. Total sequences for class A, B, and C tet genes demonstrate that their products have a common ancestor, with Tet(A) and Tet(C) being more closely related (78% identical) than either is to Tet(B) (45% identical). The N- and C-terminal halves of Tet(B) and Tet(C) appear to comprise separate domains, and trans-complementation observed between tetracycline sensitive mutants in either domain of Tet(B) suggests separate but interactive functions for these domains. In this present study, interdomain hybrid genes were constructed to express hybrid tet products whose N- and C-terminal halves were derived from different family members [Tet(A/C), Tet(B/C), and Tet(C/B)]. Tet(A/C) specified a level of Tcr comparable to wild-type Tet(C) and 60% that of Tet(A), indicating that domains from these closely related tet products can function in cis. Although neither Tet(B/C) nor Tet(C/B) hybrids conferred significant Tcr, cells producing both of these types of hybrid proteins expressed substantial Tcr, indicating that productive interactions can occur in trans between Tet(B/C) and Tet(C/B). Taken together, these results suggest that highly specific interactions between the N- and C-terminal domains are necessary for Tcr and do not occur in individual hybrids derived from the more distant relatives, Tet(B) and Tet(C). This requirement for specific interactions suggests that N- and C-terminal domains have coevolved in each member of the Tet family.
Secretion and expression of the Pasteurella haemolytica Leukotoxin
Authors: Highlander S K , Engler M J , Weinstock G M
Published: November 2016

The Pasteurella haemolytica leukotoxin gene cluster (lktCABD) is homologous to the Escherichia coli hemolysin locus (hlyCABD). Since the cloned leukotoxin (LktA) is not secreted from E. coli cells, a heteroplasmid complementation system was developed that permits secretion of the leukotoxin from cells expressing the hemolysin transport proteins HlyB and HlyD. We observed that the secreted leukotoxin protein had weak hemolytic activity when activated by either the HlyC or LktC proteins and that LktC expressed in E. coli could confer weak hemolytic activity upon hemolysin. Thus, it appears that the accessory proteins of the leukotoxin and hemolysin gene clusters are functionally similar, although their expression in E. coli is not equivalent. Northern (RNA) blot analysis of the P. haemolytica leukotoxin gene cluster revealed a major 3.5-kilobase transcript that includes the lktC and lktA genes. The start site for this transcript mapped to a cytosine residue 30 nucleotides upstream from the putative start of lktC; a similar initiation site was observed in E. coli, although adjacent cytosine and adenine residues were also utilized. The 3.5-kilobase transcript terminated near the rho-independent terminator structure between lktA and lktB, but transcription may continue, via antitermination or de novo transcription initiation, into the downstream lktB and lktD genes. We propose that the lack of LktB and LktD function in E. coli is a result, at least in part, of poor lktBD transcription and suggest that a P. haemolytica-specific regulator is required for optimal expression of the leukotoxin genes.
Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4
Authors: Perkins E J , Gordon M P , Caceres O , Lurquin P F
Published: November 2016

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorphenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro- and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and tfdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and clcD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.
Organization of a cluster of erythromycin genes in Saccharopolyspora erythraea
Authors: Weber J M , Leung J O , Maine G T , Potenz R H , Paulus T J , DeWitt J P
Published: November 2016

We used a series of gene disruptions and gene replacements to mutagenically characterize 30 kilobases of DNA in the erythromycin resistance gene (ermE) region of the Saccharopolyspora erythraea chromosome. Five previously undiscovered loci involved in the biosynthesis of erythromycin were found, eryBI, eryBII, eryCI, eryCII, and eryH; and three known loci, eryAI, eryG, and ermE, were further characterized. The new Ery phenotype, EryH, was marked by (i) the accumulation of the intermediate 6-deoxyerythronolide B (DEB), suggesting a defect in the operation of the C-6 hydroxylase system, and (ii) a block in the synthesis or addition reactions for the first sugar group. Analyses of ermE mutants indicated that ermE is the only gene required for resistance to erythromycin, and that it is not required for production of the intermediate erythronolide B (EB) or for conversion of the intermediate 3-alpha-mycarosyl erythronolide B (MEB) to erythromycin. Mutations in the eryB and eryC loci were similar to previously reported chemically induced eryB and eryC mutations blocking synthesis or attachment of the two erythromycin sugar groups. Insertion mutations in eryAI, the macrolactone synthetase, defined the largest (at least 9-kilobase) transcription unit of the cluster. These mutants help to define the physical organization of the erythromycin gene cluster, and the eryH mutants provide a source for the production of the intermediate DEB.
The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region
Authors: Whitehead T R , Hespell R B
Published: November 2016

Genes coding for three xylan-degrading activities, xylanase, xylosidase, and arabinosidase, were simultaneously cloned from the colonic anaerobic organism Bacteriodes ovatus. The genes for the three enzymes were located on a 3.8-kilobase EcoRI genomic insert and were cloned by using plasmid pUC18. All three activities were expressed in Escherichia coli JM83, and all were cell associated. Expression of the xylanase gene was independent from expression of the xylosidase and arabinosidase genes, whereas expression of the latter two genes appeared to be coordinated. Restriction endonuclease analysis of the arabinosidase and xylosidase genes and partial purification of these enzyme activities from E. coli suggested that these activities were catalyzed by a bifunctional protein or two proteins of very similar molecular weight. All three enzyme activities were regulated in B. ovatus in response to the carbon source used for growth. This is the first report of the cloning and expression of B. ovatus genes.
Rhizobium meliloti glutamate synthase: cloning and initial characterization of the glt locus
Authors: Lewis T A , Gonzalez R , Botsford J L
Published: November 2016

The genetic locus glt, encoding glutamate synthase from Rhizobium meliloti 1021, was selected from a pLAFR1 clone bank by complementation of the R. meliloti 41 Glt- mutant AK330. A fragment of cloned DNA complementing this mutant also served to complement the Escherichia coli glt null mutant PA340. Complementation studies using these mutants suggested that glutamate synthase expression requires two complementation groups present at this locus. Genomic Southern analysis using a probe of the R. meliloti 1021 glt region showed a close resemblance between R. meliloti 1021, 41, and 102f34 at glt, whereas R. meliloti 104A14 showed many differences in restriction fragment length polymorphism patterns at this locus. R. meliloti 102f34, but not the other strains, showed an additional region with sequence similarity to glt. Insertion alleles containing transposable kanamycin resistance elements were constructed and used to derive Glt- mutants of R. meliloti 1021 and 102f34. These mutants were unable to assimilate ammonia and were Nod+ Fix+ on alfalfa seedlings. The mutants also showed poor or no growth on nitrogen sources such as glutamate, aspartate, arginine, and histidine, which are utilized by the wild-type parental strains. Strains that remained auxotrophic but grew nearly as well as the wild type on these nitrogen sources were readily isolated from populations of glt insertion mutants, indicating that degradation of these amino acids is negatively regulated in R. meliloti as a result of disruptions of glt.
Transcriptional induction of an Agrobacterium regulatory gene at tandem promoters by plant-released phenolic compounds, phosphate starvation, and acidic growth media
Authors: Winans S C
Published: November 2016

Transcription of the virG gene of Agrobacterium tumefaciens was previously shown to be expressed from two tandem promoters and to be responsive to three stimuli: plant-released phenolic compounds, phosphate starvation, and acidic media. In this report, I describe a set of deletions and other alterations of the 5' end of virG that show that the upstream promoter (P1) is necessary for induction by phenolic compounds and by phosphate starvation, whereas the downstream promoter (P2) is induced by acidic media. Upstream of promoter P1 there are three copies of a family of sequences (vir boxes) found near all VirA, VirG-inducible promoters. Site-directed mutagenesis of these sequences showed that vir box I and vir box III but not vir box II are needed for induction of P1 by acetosyringone. Induction of P1 by phosphate starvation requires vir box III (or an overlapping site), whereas vir box I and vir box II are not needed. The relative importance of promoters P1 and P2 in vir gene induction was tested by measuring the expression of a virB::lacZ fusion in strains containing mutations at either promoter P1 or P2. Mutations in either promoter significantly attenuated the expression of virB, indicating that both promoters play important roles in vir gene induction.
Sequence homology between the subunits of two immunologically and functionally distinct types of fimbriae of Actinomyces spp
Authors: Yeung M K , Cisar J O
Published: November 2016

Nucleotide sequencing of the type 1 fimbrial subunit gene of Actinomyces viscosus T14V revealed a consensus ribosome-binding site followed by an open reading frame of 1,599 nucleotides. The encoded protein of 533 amino acids (Mr = 56,899) was predominantly hydrophilic except for an amino-terminal signal peptide and a carboxy-terminal region identified as a potential membrane-spanning segment. Edman degradation of the cloned protein expressed in Escherichia coli and the type 1 fimbriae of A. viscosus T14V showed that both began with alanine at position 31 of the deduced amino acid sequence. The amino acid compositions of the cloned protein and fimbriae also were comparable and in close agreement with the composition of the deduced protein. The amino acid sequence of the A. viscosus T14V type 1 fimbrial subunit showed no significant global homology with various other proteins, including the pilins of gram-negative bacteria. However, 34% amino acid sequence identity was noted between the type 1 fimbrial subunit of strain T14V and the type 2 fimbrial subunit of Actinomyces naeslundii WVU45 (M. K. Yeung and J. O. Cisar, J. Bacteriol. 170:3803-3809, 1988). This homology included several different conserved sequences of up to eight identical amino acids that were distributed in both the amino- and carboxy-terminal thirds of each Actinomyces fimbrial subunit. These findings indicate that the different types of fimbriae on these gram-positive bacteria share a common ancestry.