bluuebunny/crossref_metadata_2025 huggingface dataset:
The replication of an IncL/M plasmid is subject to antisense control
Authors: Athanasopoulos V , Praszkier J , Pittard A J
Published: November 2016

A 2,385-bp sequence that contains the information for the autonomous replication of the IncL/M plasmid pMU604 was characterized. Genetic analyses revealed that the replicon specifies at least four structural genes, designated repA, repB, repC, and rnaI. The repA gene encodes a protein with a molecular weight of 40,861 which probably functions as an initiator for replication. The functions of the proteins of the repB and repC genes are unclear; however, mutations in the start codon of repB reduced the expression of both repB and repA, indicating that these two genes are translationally coupled. The rnal gene encodes a small antisense RNA of about 75 to 77 bases and is responsible for the incompatibility phenotype, thus implicating its role as the main copy number determinant. RNAI exerts its effect in trans to repress the expression of repA at the posttranscriptional level. Furthermore, two complementary sequences of 8 bases, with the potential to interact and form a putative pseudoknot structure, were identified in the leader region of the repA mRNA. Base-pairing between the two complementary sequences was shown to be critical for efficient repA expression. A model for the regulation of pMU604 replication involving both translational coupling and pseudoknot formation is proposed.
Identification of a methyl-specific restriction system mediated by a conjugative element from Streptomyces bambergiensis
Authors: Zotchev S B , Schrempf H , Hutchinson C R
Published: November 2016

pBL2 was identified genetically but not physically in Streptomyces lividans after its mating with S. bambergiensis. During conjugation, pBL2 was transferred at high frequency to S. lividans and S. coelicolor. pBL2.1 DNA isolated from S. coelicolor exconjugants as a circular plasmid was shown to derive from the genome of S. bambergiensis. S. lividans carrying pBL2 or pBL2.1 acquired a methyl-specific restriction (MsrA+) phenotype. The corresponding enzyme was partially purified and shown to resemble a class II endonuclease which cleaves Dam-methylated DNA preferentially.
Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations
Authors: Finberg K E , Muth T R , Young S P , Maken J B , Heitritter S M , Binns A N , Banta L M
Published: November 2016

The eleven predicted gene products of the Agrobacterium tumefaciens virB operon are believed to form a transmembrane pore complex through which T-DNA export occurs. The VirB10 protein is required for virulence and is a component of an aggregate associated with the membrane fraction of A. tumefaciens. Removal of the putative membrane-spanning domain (amino acids 22 through 55) disrupts the membrane topology of VirB10 (J. E. Ward, E. M. Dale, E. W. Nester, and A. N. Binns, J. Bacteriol. 172:5200-5210, 1990). Deletion of the sequences encoding amino acids 22 to 55 abolishes the ability of plasmid-borne virB10 to complement a null mutation in the virB10 gene, suggesting that the proper topology of VirB10 in the membrane may indeed play a crucial role in T-DNA transfer to the plant cell. Western blot (immunoblot) analysis indicated that the observed loss of virulence could not be attributed to a decrease in the steady-state levels of the mutant VirB10 protein. Although the deletion of the single transmembrane domain would be expected to perturb membrane association, VirB10 delta 22-55 was found exclusively in the membrane fraction. Urea extraction studies suggested that this membrane localization might be the result of a peripheral membrane association; however, the mutant protein was found in both inner and outer membrane fractions separated by sucrose density gradient centrifugation. Both wild-type VirB10 and wild-type VirB9 were only partially removed from the membranes by extraction with 1% Triton X-100, while VirB5 and VirB8 were Triton X-100 soluble. VirB11 was stripped from the membranes by 6 M urea but not by a more mild salt extraction. The fractionation patterns of VirB9, VirB10, and VirB11 were not dependent on each other or on VirB8 or VirD4. The observed tight association of VirB9, VirB10, and VirB11 with the membrane fraction support the notion that these proteins may exist as components of multiprotein pore complexes, perhaps spanning both the inner and outer membranes of Agrobacterium cells.
Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010
Authors: Binns A N , Beaupré C E , Dale E M
Published: November 2016

The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.
Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum
Authors: Delgado M J , Yeoman K H , Wu G , Vargas C , Davies A E , Poole R K , Johnston A W , Downie J A
Published: November 2016

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.
DNA binding sites of the LysR-type regulator GcvA in the gcv and gcvA control regions of Escherichia coli
Authors: Wilson R L , Urbanowski M L , Stauffer G V
Published: November 2016

The GcvA protein is a LysR family regulatory protein necessary for both activation and repression of the Escherichia coli glycine cleavage enzyme operon (gcv) and negative regulation of gcvA. Gel shift assays indicated that overexpressed GcvA in crude extracts is capable of binding specifically to DNA containing the gcv and gcvA control regions. DNase I footprint analysis of the gcvA control region revealed one region of GcvA-mediated protection overlapping the transcription initiation site and extending from -28 to +20. Three separate GcvA binding sites in gcv were identified by DNase I footprint analysis: a 29-bp region extending from positions -271 to -242, a 28-bp region extending from -242 to -214, and a 35-bp region covering positions -69 to -34 relative to the transcription initiation site. PCR-generated mutations in any of the three GcvA binding sites in gcv decreased GcvA-mediated activation and repression of gcv.
Structure of peptidoglycan from Thermus thermophilus HB8
Authors: Quintela J C , Pittenauer E , Allmaier G , Arán V , de Pedro M A
Published: November 2016

The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported. The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide. In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid. This is the first time phenylacetic acid is described as a component of bacterial murein. Possible implications for murein physiology and biosynthesis are discussed. Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges. Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues. Neither reducing sugars nor murein-bound macromolecules were detected. Murein from T. thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms. The murein composition and peptide cross-bridges of T. thermophilus are typical for a gram-positive bacterium. However, the murein content, degree of cross-linkage, and glycan chain length for T. thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.
Use of a novel mobilizable vector to inactivate the scrA gene of Streptococcus sobrinus by allelic replacement
Authors: Buckley N D , Lee L N , LeBlanc D J
Published: November 2016

The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coli-Streptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM beta 1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(-). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289 delta 202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289 delta 202 were mobilizable by pAM beta 1 into S. sobrinus, with frequencies of 3 x 10(-6) and 1 x 10(-7) transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EIISuc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289 delta 202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM beta 1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in double crossovers was confirmed by phosphotransferase system assays. This is the first report of allelic replacement in S. sobrinus.
Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1
Authors: Ni S , Fredrickson J K , Xun L
Published: November 2016

Although reductive dehalogenation by anaerobic microorganisms offers great potential for the degradation of halocarbons, little is known about the biochemical mechanisms involved. It has previously been demonstrated that the dehalogenase activity involved in 3-chlorobenzoate dehalogenation by Desulfomonile tiedjei DCB-1 is present in the membrane fraction of the cell extracts. We report herein the purification of a 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of D. tiedjei DCB-1. The dehalogenase activity was monitored by the conversion of 3-chlorobenzoate to benzoate with reduced methyl viologen as a reducing agent. The membrane fraction of the cell extracts was obtained by ultracentrifugation, and the membrane proteins were solubilized with either the detergent CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) or Triton X-100 in the presence of glycerol. The solubilized dehalogenase was purified by ammonium sulfate fractionation and a combination of anion exchange, hydroxyapatite, and hydrophobic interaction chromatographies. This procedure yielded about 7% of the total dehalogenase activity with a 120-fold increase in specific activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified dehalogenase consisted of two subunits with molecular weights of 64,000 and 37,000. The enzyme converted 3-chlorobenzoate to benzoate at its highest specific activity in 10 mM potassium phosphate buffer (pH 7.2) at 38 degrees C. The enzyme was yellow and probably a heme protein. The enzyme had an adsorbance peak at 408 nm. The dithionite-reduced enzyme displayed absorbance peaks at 416, 522, and 550 nm. The dithionite-reduced enzyme was able to complex with carbon monoxide. The nature of the heme chromophore is currently unknown.
A novel aerobic respiratory chain-linked NADH oxidase system in Zymomonas mobilis
Authors: Kim Y J , Song K B , Rhee S K
Published: November 2016

Membrane vesicles prepared from Zymomonas mobilis oxidized NADH exclusively, whereas deamino-NADH was little oxidized. In addition, the respiratory chain-linked NADH oxidase system exhibited only a single apparent Km value of approximately 66 microM for NADH. The NADH oxidase was highly sensitive to the respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide. However, the NADH:quinone oxidoreductase was not sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide and was highly resistant to another respiratory chain inhibitor, rotenone. Electron transfer from NADH to oxygen generated a proton electrochemical gradient (inside positive) in inside-out membrane vesicles. In contrast, electron transfer from NADH to ubiquinone-1 generated no electrochemical gradient. These findings indicate that Z. mobilis possesses only NADH:quinone oxidoreductase lacking the energy coupling site.
Effects of temperature and of heat shock on the expression and action of the colicin A lysis protein
Authors: Cavard D
Published: November 2016

At low temperature, the synthesis of the colicin A lysis protein in Escherichia coli was slowed down, and consequently its functioning was retarded. The rates were restored when the bacteria were shifted for 10 min to 42 degrees C, except in an rpoH mutant, suggesting that one or more proteins regulated by sigma 32 is necessary for expression of colicin A lysis protein.
Stress response in Lactococcus lactis: cloning, expression analysis, and mutation of the lactococcal superoxide dismutase gene
Authors: Sanders J W , Leenhouts K J , Haandrikman A J , Venema G , Kok J
Published: November 2016

In an analysis of the stress response of Lactococcus lactis, three proteins that were induced under low pH culture conditions were detected. One of these was identified as the lactococcal superoxide dismutase (SodA) by N-terminal amino acid sequence analysis. The gene encoding this protein, designated sodA, was cloned by the complementation of a sodA sodB Escherichia coli strain. The deduced amino acid sequence of L. lactis SodA showed the highest degree of similarity to the manganese-containing Sod (MnSod) of Bacillus stearothermophilus. A promoter upstream of the sodA gene was identified by primer extension analysis, and an inverted repeat surrounding the -35 hexanucleotide of this promoter is possibly involved in the regulation of the expression of sodA. The expression of sodA was analyzed by transcriptional fusions with a promoterless lacZ gene. The induction of beta-galactosidase activity occurred in aerated cultures. Deletion experiments revealed that a DNA fragment of more than 130 bp surrounding the promoter was needed for the induction of lacZ expression by aeration. The growth rate of an insertion mutant of sodA did not differ from that of the wild type in standing cultures but was decreased in aerated cultures.
Steryl glycosides: a characteristic feature of the Helicobacter spp.?
Authors: Haque M , Hirai Y , Yokota K , Oguma K
Published: November 2016

The lipids of different species of Helicobacter (H.felis, H. muridarum, H. mustelae, H. fennelliae, and H. cinaedi) were studied. Different types of cholesteryl glucosides were found in all of the species studied except H. cinaedi. The total amount of cholesteryl glucosides varied from 14.8% of total lipids in H. mustelae to 33.1% of total lipids in H. felis. The different types of cholesteryl glucosides and their species distribution are cholesteryl-6-O-acyl-alpha-D-glucopyranoside (cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside in H. felis and cholesteryl-6-O-dodecanoyl-alpha-D-glucopyranoside in H. muridarum), cholesteryl-alpha-D-glucopyranoside (H. felis, H. muridarum, H. mustelae, and H. fennelliae), and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside (H. fennelliae). The neutral lipid fractions showed a high percentage of cholesterol, with selective accumulation of free cholesterol. The study thus shows that the characteristic presence of steryl glycosides in Helicobacter spp. may be an important chemotaxonomic marker for many of the species, and the helicobacters show a selective accumulation of free cholesterol from the media.
Properties of peptide chain release factor 2 from Streptomyces coelicolor A3(2): conserved primary structure but no frameshift regulation
Authors: Ogawara H , Urabe H , Ohtaki R , Nakamura Y
Published: November 2016

A gene was cloned from Streptomyces coelicolor A3(2). It encodes a protein of 368 amino acid residues with a high degree of similarity to prokaryotic release factor 2. However, it has neither an internal stop codon nor the Shine-Dalgarno-like sequence immediately upstream of the assumed frameshift position. The gene is expressed and functional in Escherichia coli as peptide chain release factor 2. The transcription start site is at or adjacent to the translational start site. The size of the mRNA detected by hybridization suggests that the gene (prfB) is monocistronic in S. coelicolor A3(2). However, about 80 bp upstream of the gene there is an operon which is composed of two genes encoding eukaryotic-type serine/threonine kinases.
Cloning of the mgtE Mg2+ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria
Authors: Townsend D E , Esenwine A J , George J , Bross D , Maguire M E , Smith R L
Published: November 2016

The MM281 strain of Salmonella typhimurium possesses mutations in each of its three Mg2+ transport systems, requires 100 mM Mg2+ for growth, and was used to screen a genomic library from the gram-negative bacterium Providencia stuartii for clones that could restore the ability to grow without Mg2+ supplementation. The clones obtained also conferred sensitivity to Co2+, a phenotype similar to that seen with the S. typhimurium corA Mg2+ transport gene. The sequence of the cloned P. stuartii DNA revealed the presence of a single open reading frame, which was shown to express a protein with a gel molecular mass of 37 kDa in agreement with the deduced size of 34 kDa. Despite a phenotype similar to that of corA and the close phylogenetic relationship between P. stuartii and S. typhimurium, this new putative Mg2+ transporter lacks similarity to the CorA Mg2+ transporter and is instead homologous to MgtE, a newly discovered Mg2+ transport protein from the gram-positive bacterium Bacillus firmus OF4. The distribution of mgtE in bacteria was studied by Southern blot hybridization to PCR amplification products. In contrast to the ubiquity of the corA gene, which encodes the dominant constitutive Mg2+ influx system of bacteria, mgtE has a much more limited phylogenetic distribution.
Genetic analysis suggests functional interactions between the N- and C-terminal domains of the TetA(C) efflux pump encoded by pBR322
Authors: McNicholas P , McGlynn M , Guay G G , Rothstein D M
Published: November 2016

Genetic analysis of the tetA(C) gene of pBR322 indicates the importance of two-cytoplasmic loops in the TetA(C) protein (P. McNicholas, I. Chopra, and D. M. Rothstein, J. Bacteriol. 174:7926-7933, 1992). In this study, we characterized second-site suppressor mutations that suggest a functional interaction between these two cytoplasmic regions of the protein.
The MalK protein of the ATP-binding cassette transporter for maltose of Escherichia coli is accessible to protease digestion from the periplasmic side of the membrane
Authors: Schneider E , Hunke S , Tebbe S
Published: November 2016

The ATP-hydrolyzing subunit MalK of the ATP-binding cassette transporter for maltose of Escherichia coli is demonstrated to be accessible to digestion by proteinase K in right-side-out membrane vesicles. This finding suggests a partial transmembrane orientation of the protein.
Phase variation of Enterococcus faecalis pAD1 conjugation functions relates to changes in iteron sequence region
Authors: Heath D G , An F Y , Weaver K E , Clewell D B
Published: November 2016

pAD1 (60 kb) is a conjugative, hemolysin/bacteriocin plasmid in Enterococcus faecalis. It confers a mating response to the peptide sex pheromone cAD1 produced by recipient (plasmid-free) cells, leading to highly efficient plasmid transfer in broth matings. Control of the physiological response to cAD1 can been overridden by a reversible phase variation event at frequencies on the order of 10(-4) to 10(-3) per cell per generation (L. T. Pontius and D. B. Clewell, Plasmid 26:172-185, 1991). The variant forms are designated Dryc and Dry+, which reflects the colony morphologies of cells whose conjugation functions are switched on and off, respectively. Here we show that Dryc variants exhibit a structural change in a region between repA and repB that contains two clusters of 8-bp iterons. The change involved a 31- or 32-bp increase in size of this region. In three or four independent variants examined, one of the iteron clusters increased in size from 13 to 17 iterons. When iteron DNA was placed on a multicopy plasmid and introduced into a wild-type pAD1 derivative, the Dryc phenotype was generated. Since traA, a key negative regulator of conjugation, bears several centrally located iteron-like sequences with the same orientation, we speculate that the protein(s) that normally binds iterons (possibly RepA and/or RepB) blocks traA transcription in Dryc variants.
Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species
Authors: Osterås M , Stanley J , Finan T M
Published: November 2016

Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum.
Increased structural and combinatorial diversity in an extended family of genes encoding Vlp surface proteins of Mycoplasma hyorhinis
Authors: Yogev D , Watson-McKown R , Rosengarten R , Im J , Wise K S
Published: November 2016

Variable lipoproteins (Vlp) constitute the major coat protein of Mycoplasma hyorhinis. They are products of multiple, divergent, single-copy genes organized in a chromosomal cluster. Three genes, vlpA, vlpB, and vlpC, have been previously identified in clonal isolates of M. hyorhinis SK76. Each is linked to a characteristic promoter region containing a homopolymeric tract of adenine residues [poly(A) tract], subject to hypermutation, that transcriptionally controls phase variation of vlp genes and leads to combinatorial surface mosaics of distinct Vlp products. The size of the natural vlp gene repertoire is unknown but may critically determine the degree of structural and combinatorial diversity available in this species. In this study, the vlp repertoire of M. hyorhinis GDL-1 was characterized and shown to contain three additional genes, vlpD, vlpE, and vlpF, clustered with other known vlp genes in the order 5'-vlpD-vlpE-vlpF-IS-vlpA-IS-vlpB-vlpC+ ++-3', where IS represents copies of the IS1221 element of M. hyorhinis. The 5' boundary of this expanded family was identical to that of the more limited family 5'-vlpA-IS-vlpB-vlpC-3' previously described in a clonal isolate of strain SK76. A recombinant construct containing vlpD, vlpE, and vlpF expressed antigenically distinguishable products corresponding to each gene. These genes encode characteristic C-terminal repetitive regions that are subject to size variation by insertion or deletion of intragenic repeats but maintain an extended, charged structure. Each vlp gene also contained characteristic alternative open reading frames, which provide a potential reservoir of coding sequence for Vlp diversity, possibly recruited through insertion and/or deletion mutations. These findings demonstrate a vastly expanded potential for structural diversity and combinatorial display of surface mosaics on this organism and suggest that modulation of the vlp repertoire, possibly in conjunction with mobile elements, may determine the capacity for surface variation in natural populations and laboratory strains of this mycoplasma species.
Self-electrophoresis is not the mechanism for motility in swimming cyanobacteria
Authors: Pitta T P , Berg H C
Published: November 2016

Swimming cyanobacteria do not have flagella. In principle, they could be propelled by streams of ions flowing from head to tail, i.e., by a self-electrophoretic mechanism. We have ruled out this possibility by showing that cells of a swimming Synechococcus species fail to drift in an external electric field.
Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1
Authors: Annunziato P W , Wright L F , Vann W F , Silver R P
Published: November 2016

The K1 capsular polysaccharide, a polymer of sialic acid, is an important virulence determinant of extraintestinal pathogenic Escherichia coli. The genes responsible for the synthesis and expression of the polysialic acid capsule of E. coli K1 are located on the 17-kb kps gene cluster, which is functionally divided into three regions. Central region 2 encodes proteins necessary for the synthesis, activation, and polymerization of sialic acid, while flanking regions 1 and 3 are involved in polymer transport to the cell surface. In this study, we identified two genes at the proximal end of region 2, neuD and neuB, which encode proteins with predicted sizes of 22.7 and 38.7 kDa, respectively. Several observations suggest that the neuB gene encodes sialic acid synthase. EV24, a neuB chromosomal mutant that expresses a capsule when provided exogenous sialic acid, could be complemented in trans by the cloned neuB gene. In addition, NeuB has significant sequence similarity to the product of the cpsB gene of Neisseria meningitidis group B, which is postulated to encode sialic acid synthase. We also present data indicating that neuD has an essential role in K1 polymer production. Cells harboring pSR426, which contains all of region 2 but lacks region 1 and 3 genes, produce an intracellular polymer. In contrast, no polymer accumulated in cells carrying a derivative of pSR426 lacking a functional neuD gene. Unlike strains with mutations in neuB, however, neuD mutants are not complemented by exogenous sialic acid, suggesting that NeuD is not involved in sialic acid synthesis. Additionally, cells harboring a mutation in neuD accumulated sialic acid and CMP-sialic acid. We also found no significant differences between the endogenous and exogenous sialyltransferase activities of a neuD mutant and the wild-type organism. NeuD shows significant similarity to a family of bacterial acetyltransferases, leading to the theory that NeuD is an acetyltransferase which may exert its influences through modification of other region 2 proteins.
Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli
Authors: Viswanathan V K , Green J M , Nichols B P
Published: November 2016

The metabolic fate of p-aminobenzoic acid (PABA) in Escherichia coli is its incorporation into the vitamin folic acid. PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate and is composed of two subunits, PabA and PabB. ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA. While there is much interest in the mechanism of chorismate aminations, there has been little work done on the ADC synthase reaction. We report that PabA requires a preincubation with dithiothreitol for maximal activity as measured by its ability to support the glutamine-dependent amination of chorismate by PabB. PabB glutamine enhances the protective effect of PabA. Incubation with fresh dithiothreitol reverses the inactivation of PabB. We conclude that both PabA and PabB have cysteine residues which are essential for catalytic function and/or for subunit interaction. Using conditions established for maximal activity of the proteins, we measured the Km values for the glutamine-dependent and ammonia-dependent aminations of chorismate, catalyzed by PabB alone and by the ADC synthase complex. Kinetic studies with substrates and the inhibitor 6-diazo-5-oxo-L-norleucine were consistent with an ordered bi-bi mechanism in which chorismate binds first. No inhibition of ADC synthase activity was observed when p-aminobenzoate, sulfanilamide, sulfathiazole, and several compounds requiring folate for their biosynthesis were used.
Characterization of toxin-producing cyanobacteria by using an oligonucleotide probe containing a tandemly repeated heptamer
Authors: Rouhiainen L , Sivonen K , Buikema W J , Haselkorn R
Published: November 2016

Cyanobacteria produce toxins that kill animals. The two main classes of cyanobacterial toxins are cyclic peptides that cause liver damage and alkaloids that block nerve transmission. Many toxin-producing strains from Finnish lakes were brought into axenic culture, and their toxins were characterized. Restriction fragment length polymorphism analysis, probing with a short tandemly repeated DNA sequence found at many locations in the chromosome of Anabaena sp. strain PCC 7120, distinguishes hepatotoxic Anabaena isolates from neurotoxin-producing strains and from Nostoc spp.
The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli
Authors: Lawrence J G , Roth J R
Published: November 2016

The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria.
Organization and transcriptional analysis of the Listeria phage A511 late gene region comprising the major capsid and tail sheath protein genes cps and tsh
Authors: Loessner M J , Scherer S
Published: November 2016

A511 is a broad-host-range, virulent myovirus for Listeria monocytogenes. The genes encoding major structural proteins of the capsid (cps) and tail sheath (tsh) were mapped to a 10.15-kb late gene fragment. We have determined the complete nucleotide sequence of this region and confirmed the identities of Cps (48.7 kDa) and Tsh (61.3 kDa) by N-terminal amino acid sequencing of both proteins. In addition, nine other open reading frames were identified. On the basis of amino acid sequence homologies to known phage-encoded proteins, some putative functions and locations could be assigned to some of the deduced gene products. We present evidence that the cps product is proteolytically cleaved between Lys-23 and Ser-24 to yield the 444-residue polypeptide found in the mature viral capsid. We also found that the N-terminal methionine is absent from the mature tail sheath protein. cps and tsh are late genes; mRNAs first appear 15 to 20 min after infection of L. monocytogenes. Northern (RNA) hybridizations of total late mRNA with specific oligonucleotide probes were used to determine the sizes of respective transcripts. Primer extension analyses enabled the positive identification of six late promoters, which were found to differ from those identified in the chromosome of Listeria spp. The bulk of transcripts from cps and tsh arise from two phage promoters with identical 13-nucleotide sequences (TGCTAGATTATAG [core region underlined]) in the -10 region which we speculate determines specific and timed expression of these genes. A 123-nucleotide leader sequence at the 5' end of the cps transcript was predicted to form a strong secondary structure (deltaG=-40.7 kcal [-170.3 kJ]/mol). Out results show that the strongly expressed A511 cps and tsh genes are included in two separate gene clusters and are independently regulated at the transcriptional level.
Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis
Authors: Otten S L , Liu X , Ferguson J , Hutchinson C R
Published: November 2016

The dnrQS genes from the daunorubicin producer Streptomyces peucetius were characterized by DNA sequencing, complementation analysis, and gene disruption. The dnrQ gene is required for daunosamine biosynthesis, and dnrS appears to encode a glycosyltransferase for the addition of the 2,3,6-trideoxy-3-aminohexose, daunosamine, to epsilon-rhodomycinone.
Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system
Authors: Sharma U K , Dwarakanath P , Banerjee N , Town C , Balganesh T S
Published: November 2016

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.
The cytochrome bc complex (menaquinone:cytochrome c reductase) in Bacillus subtilis has a nontraditional subunit organization
Authors: Yu J , Hederstedt L , Piggot P J
Published: November 2016

We have identified an operon in Bacillus subtilis, designated qcr, that is thought to encode a quinone: cytochrome c reductase. Northern (RNA blot) analysis suggests a tricistronic operon. The operon is located at about 200 degrees on the B. subtilis map. Disruption of the operon leads to loss of a 22-kDa cytochrome c from membrane preparations. The structure of the putative protein products of the qcr operon suggests a protein complex that is closely related to but distinct from known cytochrome bc1 and b6f complexes, which catalyze electron transfer from a quinol to a c-type cytochrome or to plastocyanin. QcrA is similar to Rieske-type iron-sulfur proteins; QcrB is similar in size and sequence to b-type cytochromes from b6f complexes; and QcrC has a novel structure that resembles a fusion of a subunit IV (found in b6f complexes) to a cytochrome c. Transcription of the operon is induced at the end of exponential growth from a sigma A-like promoter. This transition state induction appears to be dependent on the downregulation of abrB expression, which is mediated by Spo0A activation. As bacteria move from the transition state into sporulation, transcription of the operon is reduced in a sigma F-dependent manner.
Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli
Authors: Akerlund T , Nordström K , Bernander R
Published: November 2016

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.
Identification of the gonococcal glmU gene encoding the enzyme N-acetylglucosamine 1-phosphate uridyltransferase involved in the synthesis of UDP-GlcNAc
Authors: Ullrich J , van Putten J P
Published: November 2016

In searching for the gonococcal sialyltransferase gene(s), we cloned a 3.8-kb DNA fragment from gonococcus strain MS11 that hybridized with the oligonucleotide JU07, which was derived from the conserved C terminus of the sialyl motif present in mammalian sialyltransferases. Sequencing of the fragment revealed four putative open reading frames (ORFs), one of which (ORF-1) contained a partial sialyl motif including the amino acid sequence VGSKT, which is highly conserved among sialyltransferases. The gene was flanked by two inverted repeats containing the neisserial DNA uptake sequence and was preceded by a putative sigma 54 promoter. Database searches, however, revealed a high degree of homology between ORF-1 and the N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) of Escherichia coli and Bacillus subtilis and not with any known sialyltransferase. This homology was further established by the successful complementation of an orf-1 mutation by the E. coli glmU gene. Enzyme assays demonstrated that ORF-1 did not possess sialyltransferase activity but mimicked GlmU function catalyzing the conversion of N-acetylglucosamine 1-phosphate into UDP-N-acetylglucosamine, which is a key metabolite in the syntheses of lipopolysaccharide, peptidoglycan, and sialic acids.
Two different mechanisms mediate catabolite repression of the Bacillus subtilis levanase operon
Authors: Martin-Verstraete I , Stülke J , Klier A , Rapoport G
Published: November 2016

There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression. The LevR activator interacts with its target, the upstream activating sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter. Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala. In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon. In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis. The CRE sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter. However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation. Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.
Strong function-related homology between the pore-forming colicins K and 5
Authors: Pilsl H , Braun V
Published: November 2016

Sequence determination of the Escherichia coli colicin K determinant revealed identity with the E. coli colicin 5 determinant in the immunity and lysis proteins, strong homologies in the pore-forming region (93.7%) and the Tsx receptor-binding region (77%) of the colicins, and low levels of homology (20.3%) in the N-terminal region of the colicins. This latter region is responsible for the Tol-dependent uptake of colicin K and the Ton-dependent uptake of colicin 5 in the respective colicins. During evolution, the DNA encoding colicin activity and binding to the Tsx receptor was apparently recombined with two different DNA fragments that determined different uptake routes, leading to the differences observed in colicin K and colicin 5 import.
Properties of two sugar phosphate phosphatases from Streptococcus bovis and their potential involvement in inducer expulsion
Authors: Cook G M , Ye J J , Russell J B , Saier M H
Published: November 2016

Streptococcus bovis possesses two sugar phosphate phosphatases (Pases). Pase I is a soluble enzyme that is inhibited by the membrane fractions from lactose-grown cells and is insensitive to activation by S46D HPr, an analog of HPr(ser-P) of the sugar phosphotransferase system. Pase II is a membrane-associated enzyme that can be activated 10-fold by S46D HPr, and it appears to play a role in inducer expulsion.
In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein
Authors: Moscoso M , del Solar G , Espinosa M
Published: November 2016

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.