An Escherichia coli endonuclease responsible for primary cleavage of in vitro transcripts of bacteriophage T4 tRNA gene cluster

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.

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

Gene ◽

10.1016/0378-1119(80)90074-8 ◽

1980 ◽

Vol 11 (3-4) ◽

pp. 347-357 ◽

Cited By ~ 22

Author(s):

Alan J. Laird ◽

Douglas W. Ribbons ◽

Graeme C. Woodrow ◽

Ian G. Young

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Bacteriophage Mu ◽

Gene Transposition

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Mapping of transcription units in the bacteriophage T4 tRNA gene cluster

Journal of Molecular Biology ◽

10.1016/0022-2836(81)90039-5 ◽

1981 ◽

Vol 146 (4) ◽

pp. 393-412 ◽

Cited By ~ 16

Author(s):

Alexander Goldfarb ◽

Violet Daniel

Keyword(s):

Gene Cluster ◽

Trna Gene ◽

Bacteriophage T4

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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

Genes and Environment ◽

10.1186/s41021-020-00149-z ◽

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

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

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4703-4712.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4703-4712 ◽

Cited By ~ 16

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).

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

Applied and Environmental Microbiology ◽

10.1128/aem.01122-07 ◽

2007 ◽

Vol 73 (22) ◽

pp. 7400-7407 ◽

Cited By ~ 26

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.

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Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Journal of Bacteriology ◽

10.1128/jb.01792-07 ◽

2008 ◽

Vol 190 (10) ◽

pp. 3434-3443 ◽

Cited By ~ 24

Author(s):

Umender K. Sharma ◽

Dipankar Chatterji

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Rna Polymerase ◽

Bacteriophage T4 ◽

Sigma Factors ◽

E Coli ◽

Yeast Two Hybrid System ◽

Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

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Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.13.238 ◽

2017 ◽

Vol 13 ◽

pp. 2408-2415 ◽

Cited By ~ 4

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.

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