Cloning and Characterization of a Gene Cluster for Cyclododecanone Oxidation in Rhodococcus ruber SC1

ABSTRACT Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. colistrains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C11 to C15), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C5 to C7).

Download Full-text

High-yield production in E. coli and characterization of full-length functional p13II protein from human T-cell leukemia virus type 1

Protein Expression and Purification ◽

10.1016/j.pep.2020.105659 ◽

2020 ◽

Vol 173 ◽

pp. 105659

Author(s):

Elka R. Georgieva ◽

Peter P. Borbat ◽

Christina Fanouraki ◽

Jack H. Freed

Keyword(s):

Leukemia Virus ◽

High Yield ◽

Cell Leukemia Virus Type ◽

E Coli ◽

Yield Production ◽

Human T Cell ◽

T Cell Leukemia Virus ◽

High Yield Production

Download Full-text

Characterization of a protocatechuate catabolic gene cluster in Rhodococcus ruber OA1 involved in naphthalene degradation

Annals of Microbiology ◽

10.1007/s13213-015-1132-z ◽

2015 ◽

Vol 66 (1) ◽

pp. 469-478 ◽

Cited By ~ 8

Author(s):

Chao Li ◽

Chunyang Zhang ◽

Guanling Song ◽

Hong Liu ◽

Guihua Sheng ◽

...

Keyword(s):

Gene Cluster ◽

Rhodococcus Ruber ◽

Catabolic Gene ◽

Naphthalene Degradation

Download Full-text

Characterization of an immunogenic cellulase secreted by Cryptococcus pathogens

Medical Mycology ◽

10.1093/mmy/myaa012 ◽

2020 ◽

Vol 58 (8) ◽

pp. 1138-1148

Author(s):

Angelina Midiri ◽

Giuseppe Mancuso ◽

Germana Lentini ◽

Agata Famà ◽

Roberta Galbo ◽

...

Keyword(s):

Species Complex ◽

Delayed Type Hypersensitivity ◽

T Helper ◽

Terminal Sequence ◽

E Coli ◽

Culture Supernatants ◽

Highly Virulent ◽

Potential Use

Abstract Members of the C. neoformans/C. gattiii species complex are an important cause of serious humans infections, including meningoencephalitis. We describe here a 45 kDa extracellular cellulase purified from culture supernatants of C. neoformans var. neoformans. The N-terminal sequence obtained from the purified protein was used to isolate a clone containing the full-length coding sequence from a C. neoformans var. neoformans (strain B-3501A) cDNA library. Bioinformatics analysis indicated that this gene is present, with variable homology, in all sequenced genomes of the C. neoformans/C. gattii species complex. The cDNA clone was used to produce a recombinant 45 kDa protein in E. coli that displayed the ability to convert carboxymethyl cellulose and was therefore designated as NG-Case (standing for Neoformans Gattii Cellulase). To explore its potential use as a vaccine candidate, the recombinant protein was used to immunize mice and was found capable of inducing T helper type 1 responses and delayed-type hypersensitivity reactions, but not immune protection against a highly virulent C. neoformans var grubii strain. These data may be useful to better understand the mechanisms underlying the ability C. neoformans/C. gattii to colonize plant habitats and to interact with the human host during infection.

Download Full-text

Purification of recombinant human N-Acetyltransferase type 1 (NAT1) expressed in E. Coli and characterization of its potential role in folate metabolism

Biochemical Pharmacology ◽

10.1016/0006-2952(95)00087-g ◽

1995 ◽

Vol 49 (12) ◽

pp. 1759-1767 ◽

Cited By ~ 62

Author(s):

A. Ward ◽

M.J. Summers ◽

E. Sim

Keyword(s):

Potential Role ◽

Folate Metabolism ◽

E Coli

Download Full-text

Isolation and Characterization of Monoclonal Antibodies Directed Against Different Epitopes of Type-1-like Fimbriae from a Multifimbriated E. coli Strain

Zentralblatt für Bakteriologie ◽

10.1016/s0934-8840(11)80099-x ◽

1990 ◽

Vol 274 (2) ◽

pp. 155-173 ◽

Cited By ~ 1

Author(s):

Birgitta Biggemann ◽

Thomas Bunse ◽

Albrecht Sachsenweger ◽

Wolfgang Opferkuch

Keyword(s):

Monoclonal Antibodies ◽

Coli Strain ◽

E Coli ◽

Isolation And Characterization

Download Full-text

Characterization of a Naphthalene Catabolic Gene Cluster and Heterologous Expression of Naphthalene Dioxygenase Genes from Rhodococcus ruber OA1

Biotechnology(Faisalabad) ◽

10.3923/biotech.2017.165.173 ◽

2017 ◽

Vol 16 (4-6) ◽

pp. 165-173

Author(s):

Ying Sun ◽

Zhenglong Wang ◽

Shuyao Zhang ◽

Xiaodan Li ◽

Huaxin Gao ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Rhodococcus Ruber ◽

Catabolic Gene ◽

Naphthalene Dioxygenase ◽

Dioxygenase Genes

Download Full-text

Ferric Enterochelin Transport in Yersinia enterocolitica: Molecular and Evolutionary Aspects

Journal of Bacteriology ◽

10.1128/jb.181.20.6387-6395.1999 ◽

1999 ◽

Vol 181 (20) ◽

pp. 6387-6395 ◽

Cited By ~ 52

Author(s):

S. Schubert ◽

D. Fischer ◽

J. Heesemann

Keyword(s):

Gene Cluster ◽

Yersinia Enterocolitica ◽

Insertional Mutagenesis ◽

Yersinia Pseudotuberculosis ◽

Functional Characterization ◽

In Vitro Transcription ◽

E Coli ◽

The Family

ABSTRACT Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of theYersinia fep-fes gene cluster orthologous to theEscherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded byfepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD,fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD orfes genes abrogates enterochelin-supported growth ofY. enterocolitica on iron-chelated media. In contrast toE. coli, the fep-fes gene cluster inY. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, andYersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the familyEnterobacteriaceae.

Download Full-text

Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system.

Journal of Bacteriology ◽

10.1128/jb.177.8.2178-2187.1995 ◽

1995 ◽

Vol 177 (8) ◽

pp. 2178-2187 ◽

Cited By ~ 77

Author(s):

N Kido ◽

V I Torgov ◽

T Sugiyama ◽

K Uchiya ◽

H Sugihara ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Transport System ◽

Atp Binding ◽

E Coli ◽

Mannosyl Transferases ◽

Atp Binding Cassette

Download Full-text

Novel Acetone Metabolism in a Propane-Utilizing Bacterium, Gordonia sp. Strain TY-5

Journal of Bacteriology ◽

10.1128/jb.01054-06 ◽

2006 ◽

Vol 189 (3) ◽

pp. 886-893 ◽

Cited By ~ 60

Author(s):

Tetsuya Kotani ◽

Hiroya Yurimoto ◽

Nobuo Kato ◽

Yasuyoshi Sakai

Keyword(s):

Gene Cluster ◽

Methyl Acetate ◽

Amino Acid Sequences ◽

Gene Products ◽

Cyclic Ketones ◽

Oxidation Activity ◽

E Coli ◽

Polycistronic Transcription ◽

Terminal Amino ◽

Induced Proteins

ABSTRACT In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane → 2-propanol → acetone → methyl acetate → acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.

Download Full-text