Nucleotide sequence of plasmid pCNB1 from comamonas strain CNB-1 reveals novel genetic organization and evolution for 4-chloronitrobenzene degradation

Abstract

The nucleotide sequence of a new plasmid pCNB1 from Comamonas sp. strain CNB-1 that degrades 4-chloronitrobenzene (4CNB) was determined. pCNB1 belongs to the IncP-1beta group and is 91,181 bp in length. A total of 95 open reading frames appear to be involved in (i) the replication, maintenance, and transfer of pCNB1; (ii) resistance to arsenate and chromate; and (iii) the degradation of 4CNB. The 4CNB degradative genes and arsenate resistance genes were located on an extraordinarily large transposon (44.5 kb), proposed as TnCNB1. TnCNB1 was flanked by two IS1071 elements and represents a new member of the composite I transposon family. The 4CNB degradative genes within TnCNB1 were separated by various truncated genes and genetic homologs from other DNA molecules. Genes for chromate resistance were located on another transposon that was similar to the Tn21 transposon of the class II replicative family that is frequently responsible for the mobilization of mercury resistance genes. Resistance to arsenate and chromate were experimentally confirmed, and transcriptions of arsenate and chromate resistance genes were demonstrated by reverse transcription-PCR. These results described a new member of the IncP-1beta plasmid family, and the findings suggest that gene deletion and acquisition as well as genetic rearrangement of DNA molecules happened during the evolution of the 4CNB degradation pathway on pCNB1.

FIG. 1.

Genetic organization (A) and G+C content (B) of pCNB1 from Comamonas sp. strain CNB-1. ORFs are numbered in series, and gene names in the backbone region are indicated. The details of loading regions are shown in Fig. 4. Average G+C contents of various regions are indicated by broken lines in panel B.

FIG. 2.

Analysis of oriV (A) and oriT (B) regions of pCNB1. (A) Triangles represent the binding sites for replication initiation protein. The two rectangles stand for GC-rich and AT-rich regions. The curve shows GC molar content variations of the oriV region, and the arrow indicates the direction of replication. (B) Pairwise alignment of oriT regions from pCNB1 (upper portion) and R751 (lower portion). The sequence in the box is recognized by TraI, and the nic site is identified by the vertical arrow. From right to left, the horizontal arrows show directions of TraK transcription, plasmid transfer, and TraJ transcription.

FIG. 3.

Features of putatively new transposons at the loading regions from pCNB1. (A) Genetic organization and repeat sequences of the Tn21-like transposon, which carries a chromate-resistant gene (ORF23/CHR). (B) Genes involved in the degradation of 4-chloronitrobenzene. (C) Schematic illustration of the novel TnCNB1 with five interrupted transposase genes. Deleted fragments are indicated by the gray hashed regions in the arrows. (D) Alignment of inverted repeat (IR) sequences flanking IS1071L and IS1071R.

FIG. 4.

Comparison of genetic organizations (A) and putative metabolic pathways (B) involved in the degradation of 4-chloronitrobenzene (upper reaction sequence of panel B) and nitrobenzene (lower reaction sequence of panel B) by Comamonas sp. strain CNB-1 and Pseudomonas putida HS12. Homologous genes are filled with the same pattern. I, choloronitrobenzene nitroreductase (CnbA/NbzA); II, hydroxylaminobenzene mutase (CnbB/NbzB); III, 5-chloro/2-aminophenol 1,6-dioxygenase (CnbCaCb/NbzCaCb); IV, aminomuconic semialdehyde dehydrogenase (CnbD/NbzD); V, aminomuconate deaminase (CnbH/NbzE); V′, 4-oxalocrotonate tautomerase (CnbG); VI, 4-oxalocrotonate decarboxylase (CnbF/NbzF); VII, 2-oxo-4-pentanoate hydratase (CnbE/NbzG). Unfilled ORFs (i.e., arrows) represent unknown functions or idle genes. Portions of ORFs that are black represent lost parts of truncated genes. The numbers between genetic clusters are the identity values of the corresponding ORFs based on amino acid sequences, and an asterisk indicates that identity was calculated from DNA sequences.