Research presented in this thesis describes the genetic make‐up of extremely stable broad host range cloning vectors and their use in the stable cloning and heterologous expression of genes and gene clusters from high G+C organisms, including Streptomycetes, in Escherichia coli K12 and other heterologous hosts.
The broad host range cloning and expression vector pPSX was completely sequenced and analysed. pPSX is 14.7kb in length and contains the fusion of two continuous segments of the parental 34kb IncW plasmid R388. Segment one contains trwA', parB, orf35, oriV/repA, resP, orf38, intI1, dhfr, orfA, qacEΔ1, sul1 and orf5’. Plasmid partitioning systems are encoded in two closely linked genes parA, parB and a cis‐acting centromere‐like site parS. Sequencing of pPSX identified a homolog of ParB, but there were no direct homologs of either ParA or ParS. The multiple cloning site (MCS) from the pUC vector constructs and the cohesive end site (COS) from the pHC79 cosmid vector were inserted into orfA. The second segment contains trwA', oriT, orf18, orf19, orf20, orf21, orf22, orf23 and orf5' resides in the opposite orientation compared with pR388. There are eight open reading frames (orfs) of unknown function. orf18, 19 and 20 show homology to the stability operon (staABC) of IncN plasmids. These genes are situated next to oriT and are most likely involved in the mobilisation of pPSX during conjugation. The putative product of orf18 shows homology to the transfer protein TraD of the naphthalene catabolic plasmid pNAH7 of Pseudomonas putida. The putative product of orf23 shares some homolog with the type I restriction/modification system methylase from Burkholderia pseudomallei. One possible explanation is that orfs21, 22 and 23 encode a type I restriction/modification addiction system. If this were the case then orfs21 and 22 would encode the restriction endonuclease (R) and specificity (S) protein. Neither of the putative products of these two orfs have any homology to known endonucleases or specificity enzymes. orf35 appears to be a transcriptional regulator and its close proximity to parB suggests that it may play a role in the regulation of plasmid partitioning. orf38 is located between a resolvase gene (resP) and an integrase gene (intlI) suggesting a role in transposition.
pPSY is a 12kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10kb. In the current study, an amylase gene (amyA) derived from the high G+C Gram‐positive streptomycete Streptomyces lividans, was amplified by PCR, cloned into pGEM‐T Easy, and sub‐cloned into the EcoRI site of pPSY. The S. lividans amyA gene was strongly expressed in E. coli K12 from its native promoter. Unlike pGEM‐T Easy, pPSY stably maintained the amyA gene without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable ampliconcloning vector for the expression of heterologous genes in E. coli K12 DH5α. This demonstrates that streptomycete genes can be cloned, stably maintained and strongly expressed from their own promoters in E. coli K12. This opens up the real possibility that other streptomycete genes and gene clusters can be expressed in E. coli K12 from their own promoters.
Actinobacteria are a large group of biologically significant bacterial genera that include Streptomyces, Mycobacterium, Nocardia, Rhodococcus and Gordonia. Genomic sequencing of actinobacteria has revealed a wealth of accessory gene clusters devoted to the catabolism of complex molecules and the synthesis of antibiotics. The cloning vector pPSX allows the stable cloning and expression of antibiotic gene clusters from high G+C Gram‐negative bacteria and individual catabolic genes from high G+C Gram‐positive Streptomycetes in E. coli K12. To extend these observations to other streptomycete genes, a cosmid clone bank of the streptomycete Lechevalieria aerocolonigenes was constructed in E. coli K12 LE392 using the stable cosmid/BAC cloning vector pPSX. Clones from this bank were examined for gene expression. A novel medium, Philip Methylene Blue Salt (PMBS) agar was developed and used to detect cosmid clones which strongly absorbed/oxidized the methylene blue. When the first of these cosmid clones, designated LACB1, was sequenced it revealed a cluster of catabolic genes. One of these genes, pcpB, encoded a pentachlorophenol‐4‐monooxygenase.
Pentachlorophenol is a toxic, man made molecule, which is used as a wood preservative and is also an unwanted by‐product of the bleaching process used in paper making. When LACB1 was introduced into E. coli K12, Ralstonia eutropha JMP228 and Pseudomonas stutzeri JMP783 it conferred increased resistance to PCP. Subcloning revealed that pcpB alone can confer resistance to PCP as well as the ability to absorb/oxidize methylene blue. Since many catabolic and antibiotic gene clusters contain oxygenase genes, PMBS agar could be used to screen cosmid/BAC libraries for catabolic and antibiotic gene clusters. The heterologous expression of the oxygenase gene may indicate that other genes in these clusters are expressed in E. coli K12 from their own promoters.
Conventional genomic sequencing and analysis has revealed that functionally related bacterial genes encoding such biologically significant traits as photosynthesis, antibiotic synthesis and pathogenicity occur in gene clusters. Ongoing genomic sequencing has uncovered a significant number of additional gene clusters of unknown function. Where these gene clusters can be cloned and expressed in E. coli K12, functional analysis is greatly accelerated. If E. coli K12 does not express the gene cluster, then other genetically amenable hosts can be used. In the present study the broad host range cloning vector pPSX was used to clone the carotenoid gene cluster from Rhodobacter sphaeroides and the violacein antitumour antibiotic gene cluster from Chromobacterium violaceum and express them in a range of heterologous hosts. The carotenoid cluster was expressed in Pseudomonas stutzeri JMP783, Paracoccus denitrificans JMP228, Agrobacterium radiobacter JMP961 and Rhodobacter sphaeroides RS7001; but not expressed in Ralstonia eutropha JMP228. While the violacein gene cluster was expressed in all heterologous hosts, including E. coli K12; with very strong expression observed in A.radiobacter and P. stutzeri. This study suggests pPSX can be used to express multi‐gene phenotypes in a wide range of genetically amenable heterologous hosts.