The Completely Sequenced Plasmid pEST4011 Contains a Novel IncP1 Backbone and a Catabolic Transposon Harboring tfd Genes for 2,4-Dichlorophenoxyacetic Acid Degradation

Abstract

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Achromobacter xylosoxidans subsp. denitrificans strain EST4002 contains plasmid pEST4011. This plasmid ensures its host a stable 2,4-D ؉ phenotype. We determined the complete 76,958-bp nucleotide sequence of pEST4011. This plasmid is a deletion and duplication derivative of pD2M4, the 95-kb highly unstable laboratory ancestor of pEST4011, and was self-generated during different laboratory manipulations performed to increase the stability of the 2,4-D ؉ phenotype of the original strain, strain D2M4(pD2M4). The 47,935-bp catabolic region of pEST4011 forms a transposon-like structure with identical copies of the hybrid insertion element IS1071::IS1471 at the two ends. The catabolic regions of pEST4011 and pJP4, the best-studied 2,4-D-degradative plasmid, both contain homologous, tfd-like genes for complete 2,4-D degradation, but they have little sequence similarity other than that. The backbone genes of pEST4011 are most similar to the corresponding genes of broad-host-range self-transmissible IncP1 plasmids. The backbones of the other three IncP1 catabolic plasmids that have been sequenced (the 2,4-D-degradative plasmid pJP4, the haloacetate-catabolic plasmid pUO1, and the atrazinecatabolic plasmid pADP-1) are nearly identical to the backbone of R751, the archetype plasmid of the IncP1 ␤ subgroup. We show that despite the overall similarity in plasmid organization, the pEST4011 backbone is sufficiently different (51 to 86% amino acid sequence identity between individual backbone genes) from the backbones of members of the three IncP1 subgroups (the ␣, ␤, and ␥ subgroups) that it belongs to a new IncP1subgroup, the ␦ subgroup. This conclusion was also supported by a phylogenetic analysis of the trfA2, korA, and traG gene products of different IncP1 plasmids.Microbial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), a xenobiotic herbicide used worldwide for almost 60 years, is a well-studied process. Various soil bacteria can use 2,4-D as a carbon and energy source. Therefore, this compound has become a model for studying the evolution and distribution of genes for the degradation of chloroaromatic compounds. A number of bacterial strains belonging to different phylogenetic groups able to mineralize this compound have been found to possess genetically and enzymatically different 2,4-D-catabolic pathways (9,10,(16)(17)(18). The best-studied 2,4-D degradation genes (located in a chromosome or a plasmid) are tfd-like (pJP4-like). The very recently sequenced 87,688-bp plasmid pJP4 (48) from Wautersia eutropha JMP134 (formerly Ralstonia eutropha) was originally isolated in Australia (8), and its tfd genes and the corresponding enzymes responsible for converting 2,4-D to 3-oxoadipate are well characterized (22,23,25,26,35,58). Besides pJP4, there are only two cases in which the DNA regions containing tfd genes for the whole 2,4-D degradation pathway have been sequenced, a chromosomal transposon-like structure (about 30 kb) from Delftia acidovor...

Amino Acids in Positions 48, 52, and 73 Differentiate the Substrate Specificities of the Highly Homologous Chlorocatechol 1,2-Dioxygenases CbnA and TcbC

“…Some of the chlorocatechol gene clusters have been found to be carried on transposons (30,40,45,63) or a phage-like element (49,55). Recent reports on the entire sequencing of two 2,4-D degradative plasmids, pJP4 and pEST4011, illustrated their genetic features of basic plasmid functions such as replication, maintenance, and transfer, shared with other broad-host-range IncP1 plasmids (59,63). These findings have revealed some of the reasons underlying the wide distribution of the gene clusters of the chlorocatechol ortho-cleavage pathway.…”

The Behavior and Significance of Degradative Plasmids Belonging to Inc Groups in <i>Pseudomonas</i> within Natural Environments and Microcosms

“…The transfer of these plasmids occurs not only among the Proteobacteria, but also between bacteria of other phyla (1). Note that many degradative plasmids belong to the IncP-1 group, and that the nucleotide sequences of many such plasmids have been determined, e.g., pA81, pADP-1, pJP4, pEST4011, and pUO1 (46,57,100,114,121). The core regions of IncP-1 group plasmids show high identity.…”

Genomic and Functional Analysis of the IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Its Transposon Tn 4655 Suggests Catabolic Gene Spread by a Tyrosine Recombinase

“…Such transfer events across taxa, and subsequent mutations and rearrangements of the catabolic genes, have contributed to the rapid adaptation of bacteria to novel chemical compounds (46,52). Recent studies have shown that genes for the degradation of xenobiotic compounds, such as atrazine (31), haloacetate (44), and 2,4-dichlorophenoxyacetate (58), are predominantly carried on the incompatibility group P-1 (IncP-1) plasmids, whereas genes that encode the degradation of natural aromatic hydrocarbons, such as phenol (41), naphthalene (10,28), and toluene/xylenes (18), are usually found on IncP-2, IncP-7, and IncP-9 plasmids (9,51). Large catabolic gene clusters are also found on mobile elements integrated into bacterial chromosomes as genomic islands or conjugative transposons (8,14,53,56).…”