We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

/pdf/one-step-inactivation-of-chromosomal-genes-in-escherichia-25avsrekpc.pdf

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.

Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

/pdf/complete-genome-sequence-and-comparative-analysis-of-the-4xl8ywmhap.pdf

Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440

Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.

Aromatic compounds of both natural and man-made sources abound in the environment. The degradation of such chemicals is mainly accomplished by microorganisms. This review provides key background information but centres on recent developments in the bacterial degradation of selected man-made aromatic compounds. An aromatic compound can only be considered to be biodegraded if the ring undergoes cleavage, and this is taken as the major criteria for inclusion in this review (although the exact nature of the enzymic ring-cleavage has not been confirmed in all cases discussed).

The biodegradation of aromatic hydrocarbons by bacteria

The genetic organization of the Pseudomonas putida plasmid pWWO-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, has been investigated by transposition mutagenesis and gene cloning. Catabolic genes were localized to two clusters, one for upper pathway (hydrocarbon leads to carboxylic acid) enzymes and the other for lower pathway (carboxylic acid leads to tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase DNA segment. The physical organization of the catabolic genes thus reflects their functional organization into two regulatory blocks. The pWWO-161 DNA fragments Sst I fragment C and fragment D were cloned in a broad host range vector to produce plasmid pKT530. This hybrid encodes toluate oxygenase and all meta cleavage pathway enzymes, and it enables P. putida mt-2 and Escherichia coli K-12 cells to grow on m-toluate as sole carbon source. The pKT530 plasmid also carries xylS (a gene whose product has been postulated to regulate expression of the lower pathway genes) and the control sequences of the pathway that interact with this product, because catechol 2,3-oxygenase synthesis is specifically induced by m-toluate in both P. putida and E. coli. Evidence is presented that suggests the promoter operator of the meta pathway gene functions less effectively with the RNA polymerase or xylS product of E. coli than with the enzyme or product of P. putida.

https://www.pnas.org/content/pnas/78/12/7458.full.pdf

Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway

Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors.

A nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties. Single specific relaxation sites were located in pSC101 and RSF1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid R6K. In all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were shown to be located near origins of vegetative replication. This result suggests a functional interaction between these two types of deoxyribonucleic acid loci, and we speculate here that application events initiated at origins of replication may constitute an integral part of the process of conjugal transfer of small plasmids among bacteria. Consistent with this proposal is the finding that inhibition of vegetative replication of the pSC101 and ColE1 plasmids results in a severe inhibition of their conjugal transfer ability.

Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid.

K.N. Timmis

Papers

Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.

Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway

Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors.

Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid.