mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

ABSTRACT mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

[1]  T. Marsh.  Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. , 1999, Current opinion in microbiology.

[2]  Wolfgang Liebl,et al.  Direct Cloning from Enrichment Cultures, a Reliable Strategy for Isolation of Complete Operons and Genes from Microbial Consortia , 2001, Applied and Environmental Microbiology.

[3]  P. Trudgill,et al.  The metabolism of cyclohexanol by Acinetobacter NCIB 9871. , 1975, European journal of biochemistry.

[4]  P. Trudgill,et al.  The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871. , 1976, European journal of biochemistry.

[5]  W. Whitman,et al.  Prokaryotes: the unseen majority. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Mihovilovic,et al.  Asymmetric Baeyer—Villiger Oxidations of 4‐Mono‐ and 4,4‐Disubstituted Cyclohexanones by Whole Cells of Engineered Escherichia coli. , 2001 .

[7]  Streptococcus pneumoniae PstS Production Is Phosphate Responsive and Enhanced during Growth in the Murine Peritoneal Cavity , 2001, Infection and Immunity.

[8]  S. Delagrave,et al.  Novel approaches for discovering industrial enzymes. , 1999, Current opinion in microbiology.

[9]  A. Pardee,et al.  Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. , 1992, Science.

[10]  W. Liesack,et al.  Novel Bacterial Lineages at the (Sub)Division Level as Detected by Signature Nucleotide-Targeted Recovery of 16S rRNA Genes from Bulk Soil and Rice Roots of Flooded Rice Microcosms , 2001, Applied and Environmental Microbiology.

[11]  J. Miron,et al.  Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins? , 2000, FEMS microbiology letters.

[12]  U. Bornscheuer.  Microbial carboxyl esterases: classification, properties and application in biocatalysis. , 2002, FEMS microbiology reviews.

[13]  S. Colonna,et al.  Enantioselective synthesis of tert-butyl tert-butanethiosulfinate catalyzed by cyclohexanone monooxygenase. , 2001, Chirality.

[14]  F. de la Cruz,et al.  pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. , 1988, Gene.

[15]  C. Walsh,et al.  Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination , 1988, Journal of bacteriology.

[16]  J. Handelsman,et al.  Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms , 2000, Applied and Environmental Microbiology.

[17]  T. C. Nesbitt,et al.  Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  N. High,et al.  The Extracytoplasmic Sigma Factor, ςE, Is Required for Intracellular Survival of Nontypeable Haemophilus influenzae in J774 Macrophages , 2002, Infection and Immunity.

[19]  J. Benen,et al.  A novel oxidoreductase family sharing a conserved FAD-binding domain. , 1998, Trends in biochemical sciences.

[20]  J. Stewart,et al.  Organic transformations catalyzed by engineered yeast cells and related systems. , 2000, Current opinion in biotechnology.

[21]  P. Trudgill,et al.  Multiple forms of cyclohexanone oxygenase from Nocardia globerula CL1. , 1976, European journal of biochemistry.

[22]  G. Gottschalk,et al.  Screening of Environmental DNA Libraries for the Presence of Genes Conferring Lipolytic Activity onEscherichia coli , 2000, Applied and Environmental Microbiology.

[23]  Dick B Janssen,et al.  Identification of a Baeyer–Villiger monooxygenase sequence motif , 2002, FEBS letters.

[24]  A. Bull,et al.  Biocatalysts for clean industrial products and processes. , 1999, Current opinion in microbiology.

[25]  Yung-Hua Li,et al.  uvrA Is an Acid-Inducible Gene Involved in the Adaptive Response to Low pH inStreptococcus mutans , 2001, Journal of bacteriology.

[26]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[27]  Q. Cheng,et al.  Cloning and Characterization of a Gene Cluster for Cyclododecanone Oxidation in Rhodococcus ruber SC1 , 2001, Journal of bacteriology.

[28]  C. Kuske,et al.  Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis , 2000, Applied and Environmental Microbiology.

[29]  G. Gottschalk,et al.  Construction of Environmental DNA Libraries inEscherichia coli and Screening for the Presence of Genes Conferring Utilization of 4-Hydroxybutyrate , 1999, Applied and Environmental Microbiology.

[30]  C. Kitts,et al.  Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. , 2001, Current issues in intestinal microbiology.

[31]  Gary S. Sayler,et al.  Optimization of Differential Display of Prokaryotic mRNA: Application to Pure Culture and Soil Microcosms , 1998, Applied and Environmental Microbiology.

[32]  T. Tokuyama,et al.  Identification of a Transcriptional Activator (ChnR) and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol Catabolic Pathway in Acinetobacter sp. Strain NCIMB 9871 and Localization of the Genes That Encode Them , 1999, Applied and Environmental Microbiology.

[33]  P. Warthoe,et al.  Restriction fragment differential display of pediocin-resistant Listeria monocytogenes 412 mutants shows consistent overexpression of a putative beta-glucoside-specific PTS system. , 2000, Microbiology.

[34]  J. Ting,et al.  Identification of novel Mycoplasma hyorhinis gene fragments by differential display analysis of co-cultures. , 2001, Journal of microbiological methods.

[35]  S. Cole,et al.  Identification of novel VirR/VirS‐regulated genes in Clostridium perfringens , 2000, Molecular microbiology.

[36]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[37]  K. Edwards,et al.  Enhanced Production of Recombinant Mycobacterium tuberculosis Antigens in Escherichia coli by Replacement of Low-Usage Codons , 2000, Infection and Immunity.

[38]  M. de Mello,et al.  Differential gene expression in response to copper in Acidithiobacillus ferrooxidans analyzed by RNA arbitrarily primed polymerase chain reaction , 2002, Electrophoresis.

[39]  A. Hiraishi,et al.  Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles , 1989, Applied and environmental microbiology.

[40]  M. Bramucci,et al.  Industrial wastewater bioreactors: sources of novel microorganisms for biotechnology. , 2000, Trends in Biotechnology.

[41]  P. Rouvière,et al.  High-density sampling of a bacterial operon using mRNA differential display. , 2001, Gene.

[42]  D. Janssen,et al.  4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds. , 2001, European journal of biochemistry.

[43]  A. Pardee,et al.  Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. , 1993, Nucleic acids research.

[44]  Arthur R. Grossman,et al.  Isolation of Regulated Genes of the CyanobacteriumSynechocystis sp. Strain PCC 6803 by Differential Display , 2000, Journal of bacteriology.

[45]  Michael McClelland,et al.  Analysis of the SOS Response in Salmonella enterica Serovar Typhimurium Using RNA Fingerprinting by Arbitrarily Primed PCR , 2000, Journal of bacteriology.

[46]  Hans H. Cheng,et al.  Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA , 1997, Applied and environmental microbiology.

[47]  P. Rouvière,et al.  Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display , 2000, Journal of bacteriology.

[48]  P. Rouvière,et al.  Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacteriumepidermidis strain HCU , 2002, Applied Microbiology and Biotechnology.

[49]  J Davies,et al.  A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms , 1997, Journal of bacteriology.

[50]  J. Valentine,et al.  Genetic Analysis of a Gene Cluster for Cyclohexanol Oxidation in Acinetobacter sp. Strain SE19 by In Vitro Transposition , 2000, Journal of bacteriology.

[51]  N. Connell.  Expression systems for use in actinomycetes and related organisms. , 2001, Current opinion in biotechnology.

[52]  Michael McClelland,et al.  Arbitrarily primed PCR fingerprinting of RNA. , 1992, Nucleic acids research.

[53]  G. Muyzer,et al.  Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis , 1999, Applied and Environmental Microbiology.

[54]  D. Stahl,et al.  Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences , 1993, Applied and environmental microbiology.