Identification of a new gene, tmoF, in the Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.

Five genes, tmoABCDE, encoding toluene-4-monooxygenase (T4MO) were previously mapped to a 3.6-kb region of a 10.2-kb SacI DNA fragment isolated from Pseudomonas mendocina KR1 (K.-M. Yen, M. R. Karl, L. M. Blatt, M. J. Simon, R. B. Winter, P. R. Fausset, H. S. Lu, A. A. Harcourt, and K. K. Chen, J. Bacteriol. 173:5315-5327, 1991). In this report, we describe the identification and characterization of a DNA region in the SacI fragment whose expression enhances the T4MO activity determined by the tmoABCDE gene cluster. This region was mapped immediately downstream of the putative transcription termination sequence previously located at the end of the tmoABCDE gene cluster (Yen et al., J. Bacteriol., 1991) and was found to stimulate T4MO activity two- to threefold when expressed in Escherichia coli or Pseudomonas putida. Determination of the nucleotide sequence of this region revealed an open reading frame (ORF) of 978 bp. Expression of the ORF resulted in the synthesis of an approximately 37-kDa polypeptide whose N-terminal amino acid sequence completely matched that of the product predicted from the ORF. The ORF thus defines a gene, which has now been designated tmoF. The TmoF protein shares amino acid sequence homology with the reductases of several mono- and dioxygenase systems. In addition, the reductase component of the naphthalene dioxygenase system, encoded by the nahAa gene of plasmid NAH7 from P. putida G7, could largely replace the TmoF protein in stimulating T4MO activity, and TmoF could partially replace the NahAa protein in forming active naphthalene dioxygenase. The overall properties of tmoF suggest that it is a member of the T4mo gene cluster and encodes the NADH:ferredoxin oxidoreductase of the T4MO system.

Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.

Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The T4MO enzyme system has broad substrate specificity and provides a new opportunity for biodegradation of toxic compounds and bioconversions. Its known activities include conversion of a variety of phenyl compounds into the phenolic derivatives and the complete degradation of trichloroethylene. We have cloned and characterized a gene cluster from KR1 that determines the offO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but not on toluene. An insert consisting of two SacI fragments of identical size (10.2 kb) was shown to complement the mutant for growth on toluene. One of the SacI fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport for the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene.

Construction of cloning cartridges for development of expression vectors in gram-negative bacteria.

A cloning cartridge was constructed that can be inserted into a plasmid of choice to form an expression vector in which gene expression is inducible with an inexpensive inducer, sodium salicylate, at low concentrations. This cartridge consists of a 3.6-kb restriction fragment which contains the positive regulatory gene nahR from plasmid NAH7, a promoter, PG, that nahR regulates, a multiple cloning site, a transcription terminator, and a gene conferring tetracycline resistance. Within promoter PG of the cloning cartridge, a sequence of three nucleotides upstream of the ATG sequence encoding the initiation codon was altered to create an NdeI recognition site (CATATG) for cloning of the 5' end of a gene without affecting the distance between the transcription start site and the gene coding region. In addition, the 5' end of a gene can be converted into an NdeI recognition site without altering the amino acid sequence it encodes and then cloned into this cartridge for regulated expression. Several other synthetic restriction sites were also inserted downstream of the NdeI site for accepting the 3' end of a cloned gene. A derivative of this cloning cartridge lacking the NdeI sequence was also constructed for cloning and expression of a restriction fragment containing a gene(s) of unknown sequence. Use of the cloning cartridges in a broad-host-range plasmid has allowed successful cloning and inducible expression of several genes in all of the gram-negative bacterial tested to date. Protein production to at least 10% of the total soluble cell proteins was observed from a cloned gene expressed in Pseudomonas putida.

Genetics of naphthalene catabolism in pseudomonads.

In pseudomonads, naphthalene is catabolized in a series of reactions to salicylic acid, which is further degraded via the catechol meta-cleavage, ortho-cleavage, or gentisic acid pathway to Krebs cycle intermediates. The naphthalene catabolic genes have been located on self-transmissible plasmids, in most cases, and implicated to have chromosomal locations in other cases. The best-studied naphthalene catabolic plasmid is NAH7. It carries two operons, one of which enables the host to utilize naphthalene and the other to utilize salicylate as a carbon and energy source. The product of another NAH7 gene, nahR, is required to turn on both operons in the presence of the inducer, salicylate. Several different naphthalene and salicylate catabolic plasmids have been shown to share sequence homology with NAH7. These plasmids can undergo structural alterations involving insertions and deletions during conjugations and changes in nutritional conditions. Available evidence suggests that salicylate catabolic plasmids can form from the naphthalene catabolic plasmids by structural alterations of the plasmid DNA. The gene organization and regulation, as well as the genetic instability of the naphthalene catabolic plasmids, are reminiscent of the TOL plasmids and suggest that the naphthalene catabolic plasmids and other catabolic plasmids may have evolved in a short period of time by acquiring and modifying preevolved gene clusters from host chromosomes or other plasmids.

Regulation of naphthalene catabolic genes of plasmid NAH7.

Tn5 insertion mutations defining a regulatory gene, nahR, of the naphthalene catabolic pathway encoded by the NAH7 plasmid were mapped within a small NAH7 region only a few hundred bases upstream of the nahG gene, the most promoter-proximal gene of the nahGHIJK operon. The nahR mutations blocked the induction of both the nahABCDEF and nahGHIJK operons, and the defect was completely corrected in the presence of the wild-type allele in a trans position. The pleiotropic, recessive, and negative nature of these mutations indicates that the nahR gene specifies a regulatory element which is required to activate both nah operons.

Electron microscope heteroduplex mapping of naphthalene oxidation genes on the NAH7 and SAL1 plasmids.

Introduction of the transposon Tn5 to serve as a marker allows electron microscope heteroduplex mapping of the naphthalene oxidation genes on the approximately 83-kb NAH7 and the related approximately 85-kb SAL1 plasmids. The electron microscope-mapped gene positions on the NAH7 plasmid are in close agreement with those mapped previously by restriction digestion. The SAL1 plasmid can be considered as a mutant NAH7 plasmid which fails to direct the conversion of naphthalene to salicylate because of a mutational block but retains intact coding sequences for salicylate oxidation. Analysis of heteroduplex molecules formed between the SAL1 and NAH7::Tn5 EcoRI fragments and the known NAH7/SAL1 homology strongly suggest that the SAL1 DNA is completely homologous to NAH7 DNA except that a approximately 2.5-kb DNA segment constituting most of the nahA gene is replaced by approximately 4.6-kb nonhomologous DNA.

Plasmid gene organization: naphthalene/salicylate oxidation.

Genes for naphthalene metabolism are localized on nah7, an 83-kilobase (kb) plasmid, in two gene clusters under salicylate control. Polar mutations formed by insertion of the transposon Tn5 permit detection of the transcription direction and the gene organization within two approximately 10-kb DNA segments separated by a approximately 7-kb regulatory gene region. The gene cluster specifying conversion of naphthalene to salicylate lies near the left initiation of a 25-kb DNA fragment A released by EcoRI; that for the salicylate pathway via catechol meta-fission lies near the right terminus with extension into the adjoining 5.9-kb fragment C. The genetic organization and regulation resemble the tol plasmid-encoded "upper" and "lower" pathways of toluene/xylene oxidation in Pseudomonas putida mt2.