Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida.

Novel shuttle vectors named pEBP were constructed to allow the gene expression in different bacterial hosts including Escherichia coli, Bacillus subtilis and Pseudomonas putida. These vectors share the inducible promoters P(T7) and P(Xyl) and a cos site to enable packaging of plasmid DNA into phage, and carry different multiple cloning sites and antibiotic resistance genes. Vector pEBP41 generally replicates episomally while pEBP18 replicates episomally in Gram-negative bacteria only, but integrates into the chromosome of B. subtilis. Plasmid copy numbers determined for E. coli and P. putida were in the range of 5-50 per cell. The functionality of pEBP18 and pEBP41 was confirmed by expression of two lipolytic enzymes, namely lipase A from B. subtilis and cutinase from the eukaryotic fungus Fusarium solani pisi in three different host strains. Additionally, we report here the construction of a T7 RNA polymerase-based expression strain of P. putida.

[1]  Richard I. Gumport,et al.  Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators. , 1990, The Journal of biological chemistry.

[2]  C. Merril.  Gel-staining techniques. , 2005, Methods in enzymology.

[3]  Monica Riley,et al.  Escherichia coli K-12: a cooperatively developed annotation snapshot—2005 , 2006, Nucleic acids research.

[4]  Susan E. Cellitti,et al.  Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code , 2009, Nature Protocols.

[5]  O. White,et al.  Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. , 2002, Environmental microbiology.

[6]  Wolfgang Gärtner,et al.  Reporter proteins for in vivo fluorescence without oxygen , 2007, Nature Biotechnology.

[7]  J. Tommassen,et al.  A Novel Lipolytic Enzyme Located in the Outer Membrane of Pseudomonas aeruginosa , 1999, Journal of bacteriology.

[8]  H. Schweizer,et al.  Small broad-host-range lacZ operon fusion vector with low background activity. , 2001, BioTechniques.

[9]  L. Poulsen,et al.  New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria , 1998, Applied and Environmental Microbiology.

[10]  R. Page,et al.  Strategies to Optimize Protein Expression in E. coli , 2010, Current protocols in protein science.

[11]  M. Ikeuchi,et al.  Stable positional cloning of long continuous DNA in the Bacillus subtilis genome vector. , 2003, Journal of biochemistry.

[12]  P. Lewis,et al.  GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. , 1999, Gene.

[13]  T. Noll,et al.  Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. , 2006, Journal of molecular biology.

[14]  M. Madigan,et al.  Brock Biology of Microorganisms , 1996 .

[15]  J. Frère,et al.  Crystal structure of the Bacillus subtilis penicillin-binding protein 4a, and its complex with a peptidoglycan mimetic peptide. , 2007, Journal of molecular biology.

[16]  Stanley N Cohen,et al.  High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA , 1979, Molecular and General Genetics MGG.

[17]  C. Locht,et al.  Isolation and molecular characterization of a novel broad‐host‐range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram‐positive organisms , 1992, Molecular microbiology.

[18]  W. Quax,et al.  Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH. , 2003, FEMS microbiology letters.

[19]  T. Drepper,et al.  Novel biocatalysts for white biotechnology , 2006, Biotechnology journal.

[20]  J. Morrissey,et al.  Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems , 2011, Journal of applied microbiology.

[21]  D. Clewell,et al.  Nature of Col E1 Plasmid Replication in Escherichia coli in the Presence of Chloramphenicol , 1972, Journal of bacteriology.

[22]  F. Fuller.  A family of cloning vectors containing the lacUV5 promoter. , 1982, Gene.

[23]  Y. Koide,et al.  Characterization of pNI10 plasmid in Pseudomonas, and the construction of an improved Escherichia and Pseudomonas shuttle vector, pNUK73 , 2003, Applied Microbiology and Biotechnology.

[24]  Karl-Erich Jaeger,et al.  Novel tools for the functional expression of metagenomic DNA. , 2010, Methods in molecular biology.

[25]  B. Christensen,et al.  Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12. , 1999, Plasmid.

[26]  G. Wahl,et al.  Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Z. Su,et al.  Effect of IPTG amount on apo- and holo- forms of glycerophosphate oxidase expressed in Escherichia coli. , 2011, Protein expression and purification.

[28]  S. Ehrlich,et al.  Plasmid replication and structural stability in Bacillus subtilis. , 1991, Research in microbiology.

[29]  V. de Lorenzo,et al.  A T7 RNA polymerase-based system for the construction of Pseudomonas strains with phenotypes dependent on TOL-meta pathway effectors. , 1993, Gene.

[30]  C. Harwood,et al.  Heterologous protein secretion by bacillus species from the cradle to the grave. , 2010, Advances in applied microbiology.

[31]  T. Drepper,et al.  A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus. , 2010, Protein expression and purification.

[32]  Mapping of a transcription promoter located inside the priA gene of the Bacillus subtilis chromosome. , 2006, Acta biochimica Polonica.

[33]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[34]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[35]  J. Errington,et al.  Characterization of the essential cell division gene ftsL (yllD ) of Bacillus subtilis and its role in the assembly of the division apparatus , 1998, Molecular Microbiology.

[36]  R. Daniel,et al.  Metagenomic Analyses: Past and Future Trends , 2010, Applied and Environmental Microbiology.

[37]  C. Merril.  [36] Gel-staining techniques , 1990 .

[38]  G. del Solar,et al.  Plasmid copy number control: an ever‐growing story , 2000, Molecular microbiology.

[39]  C. J. Duggleby,et al.  Two modes of loss of the tol function from Pseudomonas putida mt-2 , 1977, Molecular and General Genetics MGG.

[40]  J. Ramos,et al.  Comparative genomics and functional analysis of niche-specific adaptation in Pseudomonas putida , 2010, FEMS microbiology reviews.

[41]  D. Chattoraj,et al.  Control of plasmid DNA replication by iterons: no longer paradoxical , 2000, Molecular microbiology.

[42]  S. Horinouchi,et al.  Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance , 1982, Journal of bacteriology.

[43]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[44]  S. S. Smith,et al.  Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. , 1989, Nucleic acids research.

[45]  S. Bron,et al.  Instability of recombinant pUB110 plasmids in Bacillus subtilis: plasmid-encoded stability function and effects of DNA inserts. , 1988, Plasmid.

[46]  A. Darzins,et al.  A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa. , 1992, Gene.

[47]  D. Dubnau,et al.  Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis , 1978, Journal of bacteriology.

[48]  M. Perego.  Integrational Vectors for Genetic Manipulation in Bacillus subtilis , 1993 .

[49]  D. Henner,et al.  Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. , 1986, Gene.

[50]  Pier Giorgio Righetti,et al.  Blue silver: A very sensitive colloidal Coomassie G‐250 staining for proteome analysis , 2004, Electrophoresis.

[51]  K. Terpe.  Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems , 2006, Applied Microbiology and Biotechnology.

[52]  N. Ravin,et al.  Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15. , 2007, Gene.

[53]  V. Neuhoff,et al.  Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250 , 1988, Electrophoresis.

[54]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[55]  M. Son,et al.  One step engineering of T7-expression strains for protein production: increasing the host-range of the T7-expression system. , 2007, Protein expression and purification.

[56]  J. Spizizen,et al.  PHYSIOLOGICAL AND GENETIC FACTORS AFFECTING TRANSFORMATION OF BACILLUS SUBTILIS , 1961, Journal of bacteriology.

[57]  Kenneth N Timmis,et al.  Pseudomonas putida: a cosmopolitan opportunist par excellence. , 2002, Environmental microbiology.

[58]  D. Ow,et al.  Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria. , 2006, Journal of microbiological methods.

[59]  G. Peterson,et al.  A simplification of the protein assay method of Lowry et al. which is more generally applicable. , 1977, Analytical biochemistry.

[60]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.

[61]  J. Frère,et al.  CENTA as a Chromogenic Substrate for Studying β-Lactamases , 2001, Antimicrobial Agents and Chemotherapy.

[62]  Germán L. Rosano,et al.  Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain , 2009, Microbial cell factories.

[63]  H. Schweizer,et al.  Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. , 2000, Plasmid.

[64]  Eduardo Díaz,et al.  Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. , 2002, Environmental microbiology.

[65]  H. Schweizer,et al.  Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. , 1994, Gene.

[66]  M. Egmond,et al.  Cutinase binding and activity at the triolein-water interface monitored by oil drop tensiometry. , 1998, Chemistry and physics of lipids.

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

[68]  O. Kuipers,et al.  Optimization of protein secretion by Bacillus subtilis. , 2008, Recent patents on biotechnology.

[69]  A. Goffeau,et al.  The complete genome sequence of the Gram-positive bacterium Bacillus subtilis , 1997, Nature.

[70]  H. Schweizer,et al.  A Tn7-based broad-range bacterial cloning and expression system , 2005, Nature Methods.

[71]  C. Anagnostopoulos,et al.  REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS , 1961, Journal of bacteriology.

[72]  S. Dunn,et al.  Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. , 1986, Analytical biochemistry.