Bacteriophage Mu-mediated gene transposition and in vitro cloning of the enterochelin gene cluster of Escherichia coli

Gene ◽  
1980 ◽  
Vol 11 (3-4) ◽  
pp. 347-357 ◽  
Author(s):  
Alan J. Laird ◽  
Douglas W. Ribbons ◽  
Graeme C. Woodrow ◽  
Ian G. Young
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Bacteriophage Mu ◽  
Gene Transposition

scholarly journals SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

2007 ◽  
Vol 282 (46) ◽  
pp. 33326-33335 ◽  
Author(s):  
David Corbett ◽  
Hayley J. Bennett ◽  
Hamdia Askar ◽  
Jeffrey Green ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Regulation Of Transcription ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Nucleoprotein Complex ◽  
Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

scholarly journals Genotyping of a gene cluster for production of colibactin and in vitro genotoxicity analysis of Escherichia coli strains obtained from the Japan Collection of Microorganisms

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Masanobu Kawanishi ◽  
Chiaki Shimohara ◽  
Yoshimitsu Oda ◽  
Yuuta Hisatomi ◽  
Yuta Tsunematsu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
In Vitro Genotoxicity

scholarly journals Proteins Encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG Genes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities

2005 ◽  
Vol 71 (8) ◽  
pp. 4703-4712 ◽  
Author(s):  
Elisabete Silva ◽  
Ana Rita Marques ◽  
Arsénio Mendes Fialho ◽  
Ana Teresa Granja ◽  
Isabel Sá-Correia
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Gelling Agent ◽  
Phosphate Binding ◽  
Sphingomonas Elodea ◽  
Gene Maps

ABSTRACT The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 μM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 μM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).

scholarly journals Analysis of the Mycosamine Biosynthesis and Attachment Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455

2007 ◽  
Vol 73 (22) ◽  
pp. 7400-7407 ◽  
Author(s):  
Aina Nedal ◽  
Håvard Sletta ◽  
Trygve Brautaset ◽  
Sven E. F. Borgos ◽  
Olga N. Sekurova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Gene Cluster ◽  
Macrolide Antibiotic ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Polyene Macrolide Antibiotic ◽  
Streptomyces Noursei ◽  
Dehydratase Activity

ABSTRACT The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the β-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

scholarly journals Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes

2017 ◽  
Vol 13 ◽  
pp. 2408-2415 ◽  
Author(s):  
Hui Hong ◽  
Markiyan Samborskyy ◽  
Katsiaryna Usachova ◽  
Katharina Schnatz ◽  
Peter F Leadlay
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Late Stage ◽  
Rare Case ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Starter Unit ◽  
Linear Polyenes ◽  
Streptomyces Malaysiensis

Clethramycin from Streptomyces malaysiensis DSM4137, and mediomycins (produced together with clethramycin from Streptomyces mediocidicus), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific O-sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene (slf) encoding a candidate sulfotransferase has been located in both gene clusters. Deletion of this gene in DSM4137 led to accumulation of desulfoclethramycin only, instead of a mixture of desulfoclethramycin and clethramycin. The mediomycin gene cluster does not encode an amidinohydrolase, but when three candidate amidinohydrolase genes from elsewhere in the S. mediocidicus genome were individually expressed in Escherichia coli and assayed, only one of them (medi4948), located 670 kbp away from the mediomycin gene cluster on the chromosome, catalysed the removal of the amidino group from desulfoclethramycin. Subsequent cloning of medi4948 into DSM4137 caused mediomycins A and B to accumulate at the expense of clethramycin and desulfoclethramycin, respectively, a rare case where an essential biosynthetic gene is not co-located with other pathway genes. Clearly, both desulfoclethramycin and clethramycin are substrates for this amidinohydrolase. Also, purified recombinant sulfotransferase from DSM4137, in the presence of 3'-phosphoadenosine-5'-phosphosulfate as donor, efficiently converted mediomycin B to mediomycin A in vitro. Thus, in the final steps of mediomycin A biosynthesis deamidination and sulfotransfer can take place in either order.

scholarly journals Mu-Like Prophage Strong Gyrase Site Sequences: Analysis of Properties Required for Promoting Efficient Mu DNA Replication

2004 ◽  
Vol 186 (14) ◽  
pp. 4575-4584 ◽  
Author(s):  
Mark Oram ◽  
Martin L. Pato
Keyword(s):  
Escherichia Coli ◽  
Dna Gyrase ◽  
Bacteriophage Mu ◽  
Reporter Plasmid ◽  
E Coli ◽  
Host Lysis ◽  
Sequences Analysis ◽  
A Site

ABSTRACT The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.

scholarly journals Characterization of Stg Fimbriae from an Avian Pathogenic Escherichia coli O78:K80 Strain and Assessment of Their Contribution to Colonization of the Chicken Respiratory Tract

2006 ◽  
Vol 188 (18) ◽  
pp. 6449-6459 ◽  
Author(s):  
Maria H. Lymberopoulos ◽  
Sébastien Houle ◽  
France Daigle ◽  
Simon Léveillé ◽  
Annie Brée ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Distinct Group ◽  
Immunogold Electron Microscopy ◽  
Carbohydrate Source ◽  
E Coli ◽  
Tract Infections ◽  
K 12

ABSTRACT In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain χ7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain χ7122 and an isogenic Δstg mutant demonstrated that compared to the wild-type parent, the Δstg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.

scholarly journals Gene Products Required for De Novo Synthesis of Polysialic Acid in Escherichia coli K1

2006 ◽  
Vol 188 (5) ◽  
pp. 1786-1797 ◽  
Author(s):  
Ekaterina N. Andreishcheva ◽  
Willie F. Vann
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
De Novo ◽  
Polysialic Acid ◽  
Neonatal Meningitis ◽  
Gene Products ◽  
De Novo Synthesis ◽  
E Coli ◽  
Tract Infections

ABSTRACT Escherichia coli K1 is responsible for 80% of E. coli neonatal meningitis and is a common pathogen in urinary tract infections. Bacteria of this serotype are encapsulated with the α(2-8)-polysialic acid NeuNAc(α2-8), common to several bacterial pathogens. The gene cluster encoding the pathway for synthesis of this polymer is organized into three regions: (i) kpsSCUDEF, (ii) neuDBACES, and (iii) kpsMT. The K1 polysialyltransferase, NeuS, cannot synthesize polysialic acid de novo without other products of the gene cluster. Membranes isolated from strains having the entire K1 gene cluster can synthesize polysialic acid de novo. We designed a series of plasmid constructs containing fragments of regions 1 and 2 in two compatible vectors to determine the minimum number of gene products required for de novo synthesis of the polysialic acid from CMP-NeuNAc in K1 E. coli. We measured the ability of the various combinations of region 1 and 2 fragments to restore polysialyltransferase activity in vitro in the absence of exogenously added polysaccharide acceptor. The products of region 2 genes neuDBACES alone were not sufficient to support de novo synthesis of polysialic acid in vitro. Only membrane fractions harboring NeuES and KpsCS could form sialic polymer in the absence of exogenous acceptor at the concentrations formed by wild-type E. coli K1 membranes. Membrane fractions harboring NeuES and KpsC together could form small quantities of the sialic polymer de novo.

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