A Restriction Map of Naphthalene Catabolic Plasmid pWW60-1 and the Location of Some of Its Catabolic Genes

Abstract

A restriction map for the 87 kbp IncP9 naphthalene catabolic plasmid pWW60-1 is presented. Transposon mutants were obtained using direct Tn.5 insertion or using an indirect method involving the intermediate formation of unstable cointegrates between RP4 and pWW60-1. Insertions which affected expression of the early enzymes of the pathway (naphthalene to salicylate) were separated from inserts which affected expression of salicylate hydroxylase (nahG) or catechol 2,3-oxygenase (nahH). nahABC were cloned on a contiguous region of the plasmid on either a 6.9 kbp Hind111 fragment (HE) or a 5.7 kbp XhoI fragment (XD). nuhGH were cloned on a region situated about 30 kbp from nahABC on an XhoI fragment (XC), but nahH was only expressed on the corresponding but smaller XhoI fragments from two derivative plasmids of pW W60-1 with a deletion in that region. The detailed restriction map of nahH shows no similarities with the restriction maps of the genes for catechol 2,3-oxygenases from TOL plasmids, although the cloned gene did hybridize with genes for catechol 2,3-oxygenase from two different TOL plasmids. The organization of the catabolic genes on pWW60-1 suggests a separation into two operons as also described for the naphthalene catabolic plasmid NAH7 (alternatively called pIG7), but the relative directions of their transcription differs from that in NAH7.

The Naphthalene Catabolic ( nag ) Genes of Ralstonia sp. Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator

“…The only other gratuitous inducer which we found was 2-nitrobenzoate, which is weakly able to induce the nag genes by a factor of two, although the level is 10-fold less than the level at which salicylate induces the operon. Much of the interest in the nag operon lies in its similarities and differences with the two other catabolic routes, (i) the classical naphthalene degradative pathway (nah) found on plasmids pDTG1 (also known as pWW60-1 [5]) and NAH7 and (ii) the nitroaromatic pathways involving an initial dioxygenase attack (see below).…”

“…strain U2 code for the enzymes of the alternative pathway for the catabolism of naphthalene, which converts naphthalene to fumarate and pyruvate via salicylate (2-hydroxybenzoate) and gentisate (2,5-dihydroxybenzoate), and they are organized in a continuous sequence of adjacent genes (8,28,29). This is in contrast to the classical naphthalene pathway enzymes, which are encoded on two separate operons, the upper-pathway operon (genes nahAa to nahF) for the conversion of naphthalene to salicylate and the lower-pathway (or meta-pathway) operon (genes nahG to nahM) for the conversion of salicylate to acetyl coenzyme A and pyruvate via catechol (5,27).…”

The 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahM and nahO genes of the naphthalene catabolic plasmid pWW60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences

“…pWW60 underwent two spontaneous structural modifications, the first during conjugational transfer from NCIMB9816 into P.ptltida PaW340, the second being a deletion which resulted in the expression of an otherwise cryptic meta pathway operon (Cane & Williams, 1982, 1986. This paper reports the cloning and functional analysis of the nahOM genes and their products from plasmid pWWG0-22.…”

Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

“…This is then converted by the action of salicylate 1-hydroxylase to catechol (1,2-dihydroxybenzene), which undergoes extradiol cleavage via the same route used for a wide range of other aromatic compounds such as toluene and the xylenes (16) and phenol (38). The nah genes are located on two separate operons: the upper pathway operon encoding the conversion of naphthalene to salicylate and the lower (or meta) pathway operon encoding the conversion of salicylate to acetyl coenzyme A and pyruvate (5,52). The induction of the two operons is linked by a common regulator protein, NahR (34).…”