bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan
Authors: Borgia P T , Dodge C L
Published: November 2016

By screening for the osmotically remediable phenotype, mutations in two genes (orlA and orlB) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, orlC and orlD, which affect cell wall beta-1,3-glucan content were also identified. Walls from strains carrying mutations in these genes exhibit normal amounts of alpha-1,3-glucan and chitin but reduced amounts of beta-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and beta-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.
mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis
Authors: Gerischer U , Dürre P
Published: November 2016

By using primer extension analysis, we located the transcription start point of the acetoacetate decarboxylase (adc) gene of Clostridium acetobutylicum 90 nucleotides upstream from the initiation codon with A as the first transcribed nucleotide. From this site the promoter structure TTTACT(18 bp)TATAAT was identified; it shows high homology to the consensus sequences of gram-positive bacteria and Escherichia coli. Northern blot experiments revealed a length of 850 bases for the transcript of the adc gene. It thus represents a monocistronic operon. Transcription of adc was induced by conditions necessary for the onset of solvent formation. Induction occurred long before the respective fermentation product (acetone) could be detected in the medium. Transcription of the operon containing the genes for acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase (designated ctf) downstream of the adc gene but divergently transcribed is also induced by conditions necessary for the onset of solvent formation. The length of the respective RNA transcript, 4.1 kb, indicates additional coding capacity, since the genes for the two subunits of the coenzyme A transferase cover only approximately 1.5 kb. No distinct transcripts for the other open reading frames of the adc gene region, ORF1 and ORF2, could be detected. Computer analysis indicated that ORF1, which showed significant similarity to the alpha-amylase gene of Bacillus subtilis (U. Gerischer and P. Dürre, J. Bacteriol. 172:6907-6918, 1990), probably is indeed a coding region. ORF2, however, does not seem to have a coding function.
Cloning and DNA sequence of a mycoplasmal recA gene
Authors: Dybvig K , Woodard A
Published: November 2016

Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.
Analysis of a mutation affecting the specificity domain for prohead binding of the bacteriophage lambda terminase
Authors: Sippy J , Feiss M
Published: November 2016

Genetic studies have identified a specificity domain for prohead binding in the C-terminal 32 amino acids of gpA, the large subunit of bacteriophage lambda terminase (S. Frackman, D. A. Siegele, and M. Feiss, J. Mol. Biol. 180:283-300, 1984). In the present work, an amber mutation, Aam42, in the fifth-to-last codon of the A gene was found to be lethal in nonsuppressing hosts. The mutation, expected to generate gpA lacking the last five amino acids, caused the production of a terminase that cut cos efficiently both in vivo and in vitro but was defective in DNA packaging. lambda Aam42 lysates contained unused proheads, consistent with a defect in prohead binding. Aam42 terminase was more strongly dependent than wild-type terminase on gpFI, the catalyst of prohead binding. Like wild-type terminase, Aam42 terminase did not cut cos in vivo when prohead assembly was blocked by a mutation in one of the genes encoding the prohead.
Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1
Authors: Southam G , Beveridge T J
Published: November 2016

Treatment of the Methanospirillum hungatei GP1 sheath with 90% (wt/vol) phenol resulted in the solubilization of a novel phenol-soluble group of polypeptides. These polypeptides were purified by the removal of insoluble material by ultracentrifugation and represented approximately 19% of the mass of the sheath. The phenol-insoluble material resembled untreated sheath but had lost its rigidity and cylindrical form. Recombination of phenol-soluble and phenol-insoluble fractions by dialysis to remove phenol resulted in cylindrical reassembly products. Although bona fide sheath (complete with the 2.8-nm lattice) was not produced, a role for the phenol-soluble polypeptides in the maintenance of sheath rigidity is implied. The phenol-soluble polypeptides have limited surface exposure as detected by antibodies on intact sheath; therefore, they are not responsible for the 2.8-nm repeat occurring on the outer face of the sheath. However, longitudinal and transverse linear labeling by protein A-colloidal gold on the outer and inner faces, respectively, occurred with monoclonal antibodies specific to the phenol-soluble polypeptides. Restricted surface exposure of phenol-soluble polypeptides on the sheath highlighted molecular defects in sheath architecture. These lattice faults may indicate sites of sheath growth to accommodate cell growth or division (longitudinal immunogold label) and filament division (transverse immunogold label). The identification of a second group of polypeptides within the infrastructure of the sheath suggests that the sheath is a trilaminar structure in which phenol-soluble polypeptides are sandwiched between sodium dodecyl sulfate-beta-mercaptoethanol-EDTA-soluble polypeptides (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) (phenol-insoluble material).
Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase
Authors: Barr G C , Bhriain N N , Dorman C J
Published: November 2016

The Escherichia coli K-12 gene coding for the nucleoid-associated protein HNS was cloned together with 5.6 kb of downstream DNA in the vector pACYC184. The cloned DNA complemented a mutation in the osmZ locus of E. coli, which codes for HNS. However, the multicopy plasmid harboring the cloned sequence was found to be mutagenic and to produce at high frequency mutations that mapped to the E. coli cya gene, which codes for adenylate cyclase. Acquisition of the cya mutations was independent of RecA. These mutations were phenotypically suppressed by providing the cells with exogenous cyclic AMP and were complemented in trans by a plasmid carrying an active copy of the cya gene. A deletion analysis of the cloned sequences showed that DNA downstream of the gene coding for HNS was also required for the mutagenic effect of cya and had a role in regulating the expression of the osmZ-dependent proU locus. These sequences appear to contain at least two genetically active regions.
A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions
Authors: Suwanto A , Kaplan S
Published: November 2016

Rhodobacter sphaeroides 2.4.1 naturally harbors five cryptic endogenous plasmids (C. S. Fornari, M. Watkins, and S. Kaplan, Plasmid 11:39-47, 1984). The smallest plasmid (pRS241e), with a molecular size of 42 kb, was observed to be a self-transmissible plasmid which can transfer only to certain strains of R. sphaeroides. Transfer frequencies can be as high as 10(-2) to 10(-3) per donor under optimal mating conditions in liquid media in the absence of oxygen. pRS241e, designated the S factor, was also shown to possess a narrow host range, failing either to replicate or to be maintained in Escherichia coli, Agrobacterium tumefaciens, and Rhizobium meliloti. It was further revealed that one of the remaining four endogenous plasmids, pRS241d, was also transmissible at a frequency similar to that of the S. factor. As a cointegrate with pSUP203, S was maintained in E. coli, providing sufficient DNA from which a physical map of S could be constructed. Progressive subcloning of S-factor DNA, in conjunction with assays of plasmid transfer, led to the localization and identification of oriV (IncA), IncB, and the putative oriT locus. The DNA sequence of the 427 bp containing oriTs revealed topological similarity to other described oriT sequences, consisting of an A-T-rich DNA region, several direct and inverted repeats, and putative integration host factor (IHF)-binding sites, and was shown to be functional in promoting plasmid transfer.
Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from "Chelatobacter" strain ATCC 29600
Authors: Uetz T , Schneider R , Snozzi M , Egli T
Published: November 2016

An assay based on the consumption of nitrilotriacetate (NTA) was developed to measure the activity of NTA monooxygenase (NTA-Mo) in cell extracts of "Chelatobacter" strain ATCC 29600 and to purify a functional, NTA-hydroxylating enzyme complex. The complex consisted of two components that easily dissociated during purification and upon dilution. Both components were purified to more than 95% homogeneity, and it was possible to reconstitute the functional, NTA-hydroxylating enzyme complex from pure component A (cA) and component B (cB). cB exhibited NTA-stimulated NADH oxidation but was unable to hydroxylate NTA. It had a native molecular mass of 88 kDa and contained flavin mononucleotide (FMN). cA had a native molecular mass of 99 kDa. No catalytic activity has yet been shown for cA alone. Under unfavorable conditions, NADH oxidation was partly or completely uncoupled from hydroxylation, resulting in the formation of H2O2. Optimum hydroxylating activity was found to be dependent on the molar ratio of the two components, the absolute concentration of the enzyme complex, and the presence of FMN. Uncoupling of the reaction was favored in the presence of high salt concentrations and in the presence of flavin adenine dinucleotide. The NTA-Mo complex was sensitive to sulfhydryl reagents, but inhibition was reversible by addition of excess dithiothreitol. The Km values for Mg(2+)-NTA, FMN, and NADH were determined as 0.5 mM, 1.3 microM, and 0.35 mM, respectively. Of 26 tested compounds, NTA was the only substrate for NTA-Mo.
Binding of Spo0A stimulates spoIIG promoter activity in Bacillus subtilis
Authors: Satola S W , Baldus J M , Moran C P
Published: November 2016

The spoIIG promoter is used by RNA polymerase containing sigma A (E sigma A), the primary form of RNA polymerase found in vegetative cells in Bacillus subtilis. However, the spoIIG promoter is active only after the onset of sporulation. Activation of the spoIIG promoter requires the product of the spo0A gene (Spo0A). Spo0A is a sequence-specific DNA-binding protein which binds to two sites in the spoIIG promoter that are essential for promoter activity. We found that single-base-pair substitutions in these two regions that reduced promoter activity in vivo caused reduced binding of Spo0A in vitro, and one substitution that increased promoter activity in vivo increased the affinity of Spo0A for this DNA in vitro. Furthermore, Spo0A stimulated transcription from the spoIIG promoter by E sigma A in vitro. These results support the model that binding of Spo0A activates E sigma A-dependent transcription from the spoIIG promoter after the onset of sporulation.
Molecular analysis of the signaling pathway between EnvZ and OmpR in Escherichia coli
Authors: Waukau J , Forst S
Published: November 2016

OmpR is a DNA-binding protein that regulates transcription of ompF and ompC. The activity of OmpR is controlled by the inner membrane osmosensor, EnvZ. In order to study the signaling process between EnvZ and OmpR, we analyzed two different envZ strains: the envZ473 strain, in which OmpC is constitutively produced and OmpF is fully repressed, and the envZ3 strain, in which the production of OmpC is greatly reduced and OmpF is not fully repressed by high-osmolarity growth conditions. Using direct sequencing of DNA derived from the polymerase chain reaction amplification method, we identified the mutation in the envZ473 strain as a Val-241-to-Gly substitution and the mutation in the envZ3 as an Ala-219-to-Val substitution. The relative DNA-binding affinity of OmpR derived from the envZ473 strain was dramatically increased for the upstream sequence of both ompF and ompC. In contrast, OmpR derived from the envZ3 strain was not converted to the high-affinity form. The intracellular levels of OmpR-phosphate, as analyzed by the in vivo phosphorylation approach, significantly increased in the envZ473 strain, while in the envZ3 strain the levels were considerably reduced, relative to those found in the parent strain. The intracellular level of OmpR protein in the envZ473 strain was also found to be markedly elevated relative to that of the parent strain. These results are discussed in relation to the role of phosphorylation and relative DNA-binding affinity of OmpR in the expression of ompF and ompC.
Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro
Authors: Pyne C , Bognar A L
Published: November 2016

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.
Physical and functional characterization of the gene cluster encoding the polyketide phytotoxin coronatine in Pseudomonas syringae pv. glycinea
Authors: Young S A , Park S K , Rodgers C , Mitchell R E , Bender C L
Published: November 2016

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The coronatine synthesis genes in PG4180 were previously shown to reside on a 90-kb plasmid designated p4180A. In the present study, clones containing a 34-kb region of p4180A were saturated with Tn5, and 71 unique mutations were recombined into p4180A by marker exchange. The effect of each mutation on coronatine synthesis was determined by analyzing the organic acids produced by the mutants by reverse-phase high-performance liquid chromatography. The organic acids of selected mutants were derivatized to their methyl esters and analyzed by gas chromatography and gas chromatography-mass spectrometry. Mutations in a 20.5-kb region of p4180A completely blocked the synthesis of coronafacic acid and coronatine. Mutations within a 4.4-kb region of p4180A prevented the formation of coronatine but allowed for production of coronafacic acid, coronafacoylvaline, coronafacoylisoleucine, and coronafacoylalloisoleucine. The phenotypes of selected mutants were further confirmed in feeding experiments in which coronafacic acid or coronamic acid was added to the culture media. The results of this study allow us to speculate on the likely sequence of steps in the later stages of coronatine biosynthesis.
Construction of Salmonella strains with both antigen O4 (of group B) and antigen O9 (of group D)
Authors: Johnson B N , Weintraub A , Lindberg A A , Stocker B A
Published: November 2016

A Salmonella live vaccine causing both O4- and O9-specific immune responses would be of use, but no reported Salmonella serotype has both of these O antigens. Constructed Salmonella typhimurium strains with an rfb (O-antigen-specifying) gene cluster of type D in the chromosome and one of type B in an F'-rfb+ factor, and those with the reverse combination reacted strongly with both anti-O4 (and anti-O5) and anti-O9 sera and, if they carried recA, could be maintained in this state by growth conditions selective for retention of the F' factor. One of the two B.rfb+ gene clusters of a (P22-lysogenic) S. typhimurium strain with a tandem duplication of a chromosomal segment including hisD and B.rfb+ was replaced (by transduction) by a D.rfb+ gene cluster; the resulting strain was O1+ O4+ O5+ O9+ and stable as such after being made recA. A stable O4+ O9+ derivative of a virulent S. enteritidis (O-group D) strain was made by transducing into it first the join point of an appropriate tandem duplication strain, together with the adjacent B.rfb+ gene cluster, and then srl::Tn10 recA.
Structural and immunochemical studies of the lipopolysaccharides of Salmonella strains with both antigen O4 and antigen O9
Authors: Weintraub A , Johnson B N , Stocker B A , Lindberg A A
Published: November 2016

Two Salmonella hybrid strains, SL5313 (Salmonella typhimurium with a D.rfb+ gene cluster) and SL5396 (S. enteritidis with a B.rfb+ gene cluster), each expressing both O-antigen 4 (of serogroup B) and O-antigen 9 (of serogroup D) were studied by immunofluorescence using a mixture of O4-specific mouse monoclonal and O9-specific rabbit polyclonal antibodies. Bound antibodies, detected by anti-mouse antibody labelled with fluorescein isothiocyanate and anti-rabbit antibody labelled with tetramethylrhodamine isothiocyanate showed that more than 98% of the bacteria expressed both the O4 and O9 epitopes. Phenol-water-extracted lipopolysaccharide from batch-grown cultures subjected to sugar and methylation analyses by gas-liquid chromatography and mass spectrometry were shown to contain abequose (of the O4 epitope) and tyvelose (of the O9 epitope) in ratios of 1:1.5 and 1:2.5 for SL5313 and SL5396, respectively. Isolated polysaccharide chains, obtained by weak-acid hydrolysis of the lipopolysaccharides, were found to contain both O4 and O9 specificities in the same molecule, since polysaccharide bound to O4 antibody attached to a solid-phase-adsorbed O9-specific antibody and vice versa. This demonstrates that in strains SL5313 and SL5396 O chains containing both O4 repeating units (from S. typhimurium) and O9 units (from S. enteritidis) are present.
Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines
Authors: Hwang I , Lim S M , Shaw P D
Published: November 2016

Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, which contained a 31-kb insert, complemented mutant NP1. A restriction map of pIH1 was constructed, and deletion analyses identified a 10-kb HindIII fragment that restored pathogenicity to NP1. Southern hybridization analysis indicated that DNA sequences in the 10-kb HindIII fragment are conserved among other X. campestris pathovars tested. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. A 2.7-kb ClaI fragment was sequenced, and two possible open reading frames (ORF1 and ORF2) were found. Results indicated that ORF2 but not ORF1 may be expressed in Escherichia coli and in X. campestris pv. glycines. The carboxy terminus of the potential polypeptide encoded by ORF2 has an amino acid sequence similar to that of the gamma subunit of oxaloacetate decarboxylase, which is involved in sodium ion transport in Klebsiella pneumoniae.
Localization and functional analysis of structural and regulatory dehalogenase genes carried on DEH from Pseudomonas putida PP3
Authors: Thomas A W , Topping A W , Slater J H , Weightman A J
Published: November 2016

Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII. The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location. From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned. Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P. putida and Escherichia coli. The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI). Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA). Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters. Expression of dehI in P. putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent. The intact DEH element transferred to P. putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA. This indicated that the cloned DEH element contained functions associated with recombination.
Cross-linkage and cross-linking of peptidoglycan in Escherichia coli: definition, determination, and implications
Authors: Driehuis F , de Jonge B , Nanninga N
Published: November 2016

The glycan chains in peptidoglycan or murein are cross-linked by transpeptidation of the peptide side chains. To assess the fraction of side chains involved in cross-bridges, distinction has been made between cross-linkage and cross-linking. The first expression refers to the situation in unlabeled (or fully labeled) peptidoglycan, and the second refers to pulse-labeled peptidoglycan. It is argued that for the determination of the cross-linking value, the mode of insertion as denoted by the so-called acceptor/donor radioactivity ratio should be taken into account.
Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogen-limiting conditions
Authors: Cussac V , Ferrero R L , Labigne A
Published: November 2016

Helicobacter pylori produces a potent urease that is believed to play a role in the pathogenesis of gastroduodenal diseases. Four genes (ureA, ureB, ureC, and ureD) were previously shown to be able to achieve a urease-positive phenotype when introduced into Campylobacter jejuni, whereas Escherichia coli cells harboring these genes did not express urease activity (A. Labigne, V. Cussac, and P. Courcoux, J. Bacteriol. 173:1920-1931, 1991). Results that demonstrate that H. pylori urease genes could be expressed in E. coli are presented in this article. This expression was found to be dependent on the presence of accessory urease genes hitherto undescribed. Subcloning of the recombinant cosmid pILL585, followed by restriction analyses, resulted in the cloning of an 11.2-kb fragment (pILL753) which allowed the detection of urease activity (0.83 +/- 0.39 mumol of urea hydrolyzed per min/mg of protein) in E. coli cells grown under nitrogen-limiting conditions. Transposon mutagenesis of pILL753 with mini-Tn3-Km permitted the identification of a 3.3-kb DNA region that, in addition to the 4.2-kb region previously identified, was essential for urease activity in E. coli. Sequencing of the 3.3-kb DNA fragment revealed the presence of five open reading frames encoding polypeptides with predicted molecular weights of 20,701 (UreE), 28,530 (UreF), 21,744 (UreG), 29,650 (UreH), and 19,819 (UreI). Of the nine urease genes identified, ureA, ureB, ureF, ureG, and ureH were shown to be required for urease expression in E. coli, as mutations in each of these genes led to negative phenotypes. The ureC, ureD, and ureI genes are not essential for urease expression in E. coli, although they belong to the urease gene cluster. The predicted UreE and UreG polypeptides exhibit some degree of similarity with the respective polypeptides encoded by the accessory genes of the Klebsiella aerogenes urease operon (33 and 92% similarity, respectively, taking into account conservative amino acid changes), whereas this homology was restricted to a domain of the UreF polypeptide (44% similarity for the last 73 amino acids of the K. aerogenes UreF polypeptide). With the exception of the two UreA and UreB structural polypeptides of the enzyme, no role can as yet be assigned to the nine proteins encoded by the H. pylori urease gene cluster.
Identification of proteins phosphorylated by ATP during sporulation of Bacillus subtilis
Authors: Mitchell C , Morris P W , Vary J C
Published: November 2016

Protein phosphorylation in Bacillus subtilis was assayed in vitro by using extracts prepared from cells at various times during growth and sporulation. At least six proteins were labeled in vitro by using [gamma-32P]ATP and extracts of vegetative cells. In extracts prepared at the end of exponential growth and during stationary phase, 12 to 13 proteins were labeled. Seven of the phosphoproteins were purified by fast-performance liquid chromatography and polyacrylamide gel electrophoresis, blotted to Immobilon membranes, and subjected to partial protein sequencing. One of the sequences had sequence homology (greater than 45%) to elongation factor G from several bacterial species, and four sequences matched the predicted amino-terminal sequences of the outB, orfY-tsr, orfU, and ptsH genes.
A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene
Authors: Sugino H , Sasaki M , Azakami H , Yamashita M , Murooka Y
Published: November 2016

The Klebsiella aerogenes gene maoA, which is involved in the synthesis of monoamine oxidase, was induced by tyramine and the related compounds, subjected to catabolite and ammonium ion repression, and cloned. The nucleotide sequence of the region involved in monoamine oxidase synthesis was determined. Two open reading frames, the maoA gene and a hitherto unknown gene (maoC), were found. These are located between a potential promoter sequence and a transcriptional terminator sequence. A region of the Escherichia coli chromosome that was highly homologous to the Klebsiella maoA gene was found. The potential maoA gene is located at 30.9 min on the E. coli chromosome. Analysis of the amino acid sequences of the first 11 amino acids from the N terminus of the purified monoamine oxidase agrees with those deduced from the nucleotide sequence of the maoA gene. The leader peptide extends over 30 amino acids and has the characteristics of a signal sequence. Primer extension and S1 nuclease mapping of transcripts generated in vivo suggests that the tyramine-induced mRNA starts at a site 62 bases upstream from the ATG initiation codon of the maoC gene. In the putative promoter region, a high degree of similarity to the consensus sequence for the binding site of cyclic AMP receptor protein was found. Thus, the mao region is composed of two cistrons, and the mao operon is regulated by monoamine compounds, glucose, and ammonium ions.
The VanS-VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147
Authors: Arthur M , Molinas C , Courvalin P
Published: November 2016

Plasmid pIP816 of Enterococcus faecium BM4147 confers inducible resistance to vancomycin and encodes the VanH dehydrogenase and the VanA ligase for synthesis of depsipeptide-containing peptidoglycan precursors which bind the antibiotic with reduced affinity. We have characterized a cluster of five genes of pIP816 sufficient for peptidoglycan synthesis in the presence of vancomycin. The distal part of the van cluster encodes VanH, VanA, and a third enzyme, VanX, all of which are necessary for resistance. Synthesis of these enzymes was regulated at the transcriptional level by the VanS-VanR two-component regulatory system encoded by the proximal part of the cluster. VanR was a transcriptional activator related to response regulators of the OmpR subclass. VanS stimulated VanR-dependent transcription and was related to membrane-associated histidine protein kinases which control the level of phosphorylation of response regulators. Analysis of transcriptional fusions with a reporter gene and RNA mapping indicated that the VanR-VanS two-component regulatory system activates a promoter used for cotranscription of the vanH, vanA, and vanX resistance genes.
Purification and partial characterization of transglutaminase from Physarum polycephalum
Authors: Klein J D , Guzman E , Kuehn G D
Published: November 2016

An intracellular form of calcium ion-dependent transglutaminase (R-glutaminylpeptide:amine gamma-glutaminyltransferase, EC 2.3.2.13) was purified 818-fold to apparent homogeneity from acetone powder preparations of spherules of the acellular slime mold Physarum polycephalum. The enzyme was purified by combined methods of precipitation with 15% (wt/vol) polyethylene glycol, DEAE-cellulose chromatography, and isoelectric focusing in a pH 5 to 7 gradient. The isoelectric point of the enzyme was 6.1. The molecular mass of the denatured enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 39.6 kDa. A molecular weight of 77,000 was found by gel filtration of the native enzyme on a Superose 12 fast protein liquid chromatography column, indicating that the native functional protein is a dimer. The purified transglutaminase catalyzed the incorporation of [14C]putrescine into protein substrates including casein, N,N'-dimethylcasein, actin purified from P. polycephalum, and actin purified from bovine muscle. Actin was the preferred substrate for the enzyme, both as a purified protein and in crude extracts prepared from P. polycephalum. With N,N'-dimethylcasein as the amine acceptor substrate, [14C]putrescine, [14C]spermidine, and [14C]spermine were all effective amine donor substrates with Km values of 49, 21.4, and 31.7 microM, respectively. All three of these polyamines demonstrated strong substrate inhibition of the enzyme activity between 100 and 200 microM. Upon starvation induced by depletion of a carbon source for growth, the specific activity of this enzyme increased sixfold during the differentiation of P. polycephalum microplasmodia to spherules. This suggests a role for transglutaminase in the construction of spherules, which have the capacity to survive starvation and dessication.
Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens
Authors: LaPointe G , Nautiyal C S , Chilton W S , Farrand S K , Dion P
Published: November 2016

Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine. The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs. [14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized [14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14C]mannopine, was in the form of agropine.
Spo0A controls the sigma A-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE
Authors: York K , Kenney T J , Satola S , Moran C P , Poth H , Youngman P
Published: November 2016

The spoIIE operon is a developmentally regulated transcription unit activated in the second hour of sporulation in Bacillus subtilis. Its promoter has an unusual structure, containing sequences which conform perfectly to the consensus for vegetative promoters recognized by sigma A-associated RNA polymerase (E sigma A), but with a spacing of 21 bp between the apparent -10 and -35 elements instead of the 17- or 18-bp spacing typical of promoters utilized by E sigma A. Mutations introduced into the apparent -10 element affected transcription in a manner consistent with its functioning as a polymerase recognition sequence. The deleterious effect of one -10 mutation was also suppressed in an allele-specific manner by a mutation in sigA known to suppress analogous -10 mutations in conventional vegetative promoters recognized by E sigma A. Similar suppression experiments failed to provide evidence for a direct interaction between E sigma A and the "-35-like" element, however, and DNase I protection experiments suggested instead that the Spo0A protein binds to a site overlapping this -35-like hexamer. Moreover, the effects of mutations within the -35-like hexamer on the binding of Spo0A in vitro paralleled their effects on transcription in vivo. We suggest that spoIIE belongs to a class of early-intermediate sporulation genes whose transcription by E sigma A is activated by the Spo0A protein.
Regulation of the Escherichia coli cad operon: location of a site required for acid induction
Authors: Meng S Y , Bennett G N
Published: November 2016

The cad operon encodes lysine decarboxylase and a protein homologous to amino acid antiporters. These two genes are induced under conditions of low pH, anaerobiosis, and excess lysine. The upstream regulatory region of the cad operon has been cloned into lacZ expression vectors for analysis of the sequences involved in these responses. Deletion analysis of the upstream region and cloning of various fragments to make cadA::lacZ or cadB::lacZ protein fusions or operon fusions showed that cadA was translated more efficiently than cadB and localized the pH-responsive site to a region near an upstream EcoRV site. Construction of defined end points by polymerase chain reaction further localized the left end of the regulatory site. The presence of short fragments bearing the regulatory region on high-copy-number plasmids greatly reduced expression from the chromosomal cad operon, suggesting that titration of an essential activator protein was occurring. With nonoptimal polymerase chain reaction conditions, a set of single point mutants were made in the upstream regulatory region. Certain of these altered regulatory regions were unable to compete for the regulatory factor in vivo. The locations of these essential bases indicate that a sequence near the EcoRV site is very important for the activator-DNA interaction. In vivo methylation experiments were conducted with cells grown at pH 5.5 or at pH 8, and a difference in protection was observed at specific G residues in and around the region defined as important in pH regulation by the mutation studies. This work defines essential sequences for acid induction of this system involved in neutralization of extracellular acid.
Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris
Authors: Hu N T , Hung M N , Chiou S J , Tang F , Chiang D C , Huang H Y , Wu C Y
Published: November 2016

Nonpathogenic mutants of Xanthomonas campestris pv. campestris, generated from transposon mutagenesis, accumulated extracellular polygalacturonate lyase, alpha-amylase, and endoglucanase in the periplasm. The transposon Tn5 was introduced by a mobilizable, suicidal plasmid, pSUP2021 or pEYDG1. Genomic banks of wild-type X. campestris pv. campestris, constructed on the broad-host-range, mobilizable cosmid pLAFR1 or pLAFR3, were conjugated with one of the mutants, designated XC1708. Recombinant plasmids isolated by their ability to complement XC1708 can be classified into two categories. One, represented by pLASC3, can complement some mutants, whereas the other, represented by a single plasmid, pLAHH2, can complement all of the other mutants. Restriction mapping showed that the two recombinant plasmids shared an EcoRI fragment of 8.9 kb. Results from subcloning, deletion mapping, and mini-Mu insertional mutation of the 8.9-kb EcoRI fragment suggested that a 4.2-kb fragment was sufficient to complement the mutant XC1708. Sequence analysis of this 4.2-kb fragment revealed three consecutive open reading frames (ORFs), ORF1, ORF2, and ORF3. Hybridization experiments showed that Tn5 in the genome of XC1708 and other mutants complemented by pLASC3 was located in ORF3, which could code for a protein of 83.5 kDa. A signal peptidase II processing site was identified at the N terminus of the predicted amino acid sequence. Sequence homology of 51% was observed between the amino acid sequences predicted from ORF3 and the pulD gene of Klebsiella species.
Diabetes and diabetic retinopathy in people aged 50 years and older in Hungary
Authors: Tóth Gábor , Szabó Dorottya , Sándor Gábor L , Szalai Irén , Lukács Regina , Pék Anita , Tóth Georgina Z , Papp András , Nagy Zoltán Z , Limburg Hans , Németh János
Published: October 2016

Background/aims

The purpose of this study was to estimate the prevalence of diabetes mellitus (DM) and diabetic retinopathy (DR) in the population aged 50 years and older in Hungary, and to assess the coverage of diabetic eye care services.

Methods

In total, 105 clusters of 35 people aged 50 years or older were randomly selected. The standardised rapid assessment of avoidable blindness (RAAB) with the diabetic retinopathy module (DRM) was used. Participants were classified as having DM if they were known to have DM or if their random blood glucose level was ≥200 mg/dL. Dilated fundus examination and Scottish DR grading were performed.

Results

In total, 3523 (95.9%) out of 3675 eligible subjects were examined. And 705 (20.0%) out of 3523 had known (661) or newly diagnosed DM (44). Twenty per cent of participants with known DM had a blood glucose level ≥200 mg/dL, and 27.4% had never had an ophthalmological examination for DR. Prevalence of DR and/or maculopathy was 20.7% and prevalence of sight-threatening DR (STDR) was 4.3% in one or both eyes among participants with DM.

Conclusions

Prevalence of DM was in line with findings of other RAAB+DRM surveys and slightly lower than the unpublished earlier age-matched Hungarian estimate. Prevalence of DR was slightly lower than expected. The prevalence of STDR was low in people aged 50 years and older in Hungary compared with the results of other RAAB with DRM surveys. DR screening coverage was low. To prevent severe complications of DM and possible concomitant visual loss, the coverage of ophthalmic examinations in patients with DM should be increased.
Natural history and effect of therapeutic interventions on subretinal fluid causing foveal detachment in macular telangiectasia type 2
Authors: Mehta Hemal , Müller Simone , Egan Catherine A , Degli Esposti Simona , Tufail Adnan , Sim Dawn A , Holz Frank G , Browning Andrew C , Amoaku Winfried M , Charbel Issa Peter , Gillies Mark C
Published: October 2016

Aim

To report the natural history of subretinal fluid (SRF) causing foveal detachment in macular telangiectasia type 2 (MacTel) and our experience of therapeutic intervention with intravitreal steroids or antivascular endothelial growth factor inhibitor (anti-VEGF) agents in some cases.

Methods

Retrospective case series. Three of the MacTel study's largest registries were searched to identify eyes with foveal detachment.

Results

We identified 7 eyes from 6 exclusively female patients. The prevalence of foveal detachment was low, present in 1.4% of the assessed MacTel population. Age at presentation ranged from 50 to 66 years. Follow-up ranged from 2 to 8 years. There was late-phase leakage on fluorescein angiography from what was presumed to be ectatic capillaries. The SRF fluctuated without a rapid decline in visual acuity in cases that were not treated. When they were, intravitreal anti-VEGF and steroid therapy in general reduced SRF, at least temporarily, but did not halt the gradual long-term decrease in visual acuity. In one case, optical coherence tomography angiography showed significant reduction in the extent of the predominantly deep intraretinal vascular complex 1 month after anti-VEGF therapy.

Discussion and conclusions

As the natural history of this unusual MacTel phenotype is not characterised by rapid visual decline, intervention with intravitreal anti-VEGF or steroid therapy may not be necessary.
Anatomical and functional outcomes following vitrectomy for advanced familial exudative vitreoretinopathy: a single surgeon's experience
Authors: Hocaoglu Mumin , Karacorlu Murat , Sayman Muslubas Isil , Ersoz Mehmet Giray , Arf Serra
Published: October 2016

Purpose

To assess the anatomical and functional results of vitreoretinal surgery in patients with advanced familial exudative vitreoretinopathy (FEVR).

Methods

Retrospective analysis of data for 10 eyes of 9 patients with advanced FEVR who underwent pars plana vitrectomy from March 1997 to May 2015 and had a follow-up of at least 12 months. The primary outcomes were final visual acuity (VA) and anatomical success.

Results

The average age at the time of the surgery was 10.1±6.5 years (range 2 months–18 years). The mean follow-up period was 58.4±75.1 months. The male-to-female ratio was 7/2. The mean number of vitreoretinal operations was 1.5. At the last visit, 7 (70%) eyes had complete or partial retinal attachment. Preoperatively, the mean Snellen VA was 20/4000 (n=8), and in the remaining 2 (20%) eyes it was recorded as ‘central, steady and maintained’. At final examination, the mean Snellen VA was 20/330 (n=7), and in the remaining 3 (30%) eyes it was recorded as light perception. At the final visit, 5 eyes (50%) had improved VA, 2 eyes (20%) showed stabilisation, and 3 eyes (30%) with total retinal detachment had a decrease in VA. No progression to glaucoma was observed and no enucleation was necessary.

Conclusions

Advanced FEVR in young children tends to be more aggressive, leading to severe complications. These cases are challenging and require special consideration. Despite surgery, disease-related complications remain high. However, surgical intervention for advanced FEVR might be of benefit in helping to preserve vision.
Educating professionals to support self-management in people with asthma or diabetes: protocol for a systematic review and scoping exercise
Authors: McCleary Nicola , Andrews Amanda , Morrow Susan , Wiener-Ogilvie Sharon , Fletcher Monica , Steed Liz , Taylor Stephanie J C , Pinnock Hilary
Published: October 2016

Introduction

Supported self-management for asthma helps people adjust their treatment in response to symptom changes. This improves day-to-day control and reduces the risk of asthma attacks and the need for emergency healthcare. However, implementation remains poor in routine clinical practice. This systematic review is part of a programme of work developing an intervention to help primary care practice teams embed self-management support into routine asthma care. The aim of the review is to synthesise the evidence regarding the effectiveness of educational interventions for professionals supporting self-management in people with asthma or diabetes (type 1 and type 2). These two conditions have the most robust evidence base for the effectiveness of implementing supported self-management.

Methods and analysis

Electronic searches will be conducted in CENTRAL, MEDLINE, EMBASE, ISI Web of Science, CINAHL, PsycINFO, AMED, Global Health, WHO Global Health Library, ERIC, BNI, RDRB/CME and Google Scholar. Eligible studies are randomised controlled trials or controlled clinical trials published between 1990 and 2016 which evaluated professional education interventions facilitating asthma or diabetes supported self-management. Further relevant work will be identified from trial registries, citation searching and through contact with authors of included studies. This will be supplemented by scoping potentially relevant educational packages described in English language policy literature or health service websites. Screening, data extraction and risk of bias assessment (using the Cochrane Risk of Bias Tool) will be completed by two independent reviewers, with a third reviewer arbitrating where necessary. We plan a theoretically informed narrative synthesis of the aggregated data as heterogeneity is likely to preclude meta-analysis.

Ethics and dissemination

Ethical approval is not required for this systematic review. The results will be described in a paper submitted for peer-reviewed publication and will inform the development of an implementation intervention.

Study registration number

PROSPERO CRD42016032922.