Plasmid pRK2013 ( 18) was used as a helper plasmid in triparental matings. SB354, a Tn 5 derivative of the suicide vector pRK600 ( 85) was maintained on LB containing kanamycin to select for the transposon and chloramphenicol to select for the pRK600 plasmid containing the transposon. Bacteria were maintained on either full-strength ( Escherichia coli) or half-strength ( Sphingomonas) Luria-Bertani medium (LB). paucimobilis ATCC 298377 was purchased from the American Type Culture Collection. Its resistance to bacitracin and inability to degrade aromatic compounds examined in this study was useful for plasmid transfer experiments. strain S-88 (ATCC 31554) mutant m260 was obtained from Thomas Pollock ( 77). Department of Energy’s Subsurface Microbial Culture Collection at Florida State University ( 3). aromaticivorans F199, originally isolated by our laboratory, is also maintained in the U.S. yanoikuyae B1, and the development of hypotheses regarding functions of pNL1-encoded genes.īacterial strains, plasmids, and culture conditions. This approach has allowed a thorough genetic analysis of pNL1-associated catabolic genes, a comparison of these genes with analogous chromosomally located ones in S. To further probe the catabolic functions and accessory genes encoded on pNL1, we undertook the complete sequencing and annotation of this plasmid. We reported earlier that catechol meta ring cleavage activity, a central step in the catabolism of aromatic rings, was associated with the smaller plasmid, pNL1 ( 86), and we described a physical map for this plasmid. aromaticivorans F199 was shown to possess two plasmids ( 20, 22), which are designated pNL1 (∼180 kbp) and pNL2 (∼480 kbp). This unusual gene arrangement suggests that a highly complex regulatory network is responsible for the expression of aromatic degradative pathways in some Sphingomonas spp. Some Sphingomonas strains are further distinguished in that the genes necessary for degradation of one type of aromatic compound are distributed into multiple operons that also possess genes for the degradation of other aromatic compounds ( 107). evolved independently from phylogenetically distinct bacteria such as those within the genus Pseudomonas ( 46, 47). The inability to detect Sphingomonas biodegradative genes via hybridization with catabolic genes from phylogenetically distinct bacteria suggested that biodegradative genes from Sphingomonas sp. Studies of Sphingomonas strains suggest that members of this genus are well adapted for the degradation of high-molecular-weight polycyclic aromatic hydrocarbons and other aromatic contaminants. ![]() ![]() In recent years, there have been many reports of other Sphingomonas strains that are capable of degrading aromatic compounds ( 12, 17, 30, 36, 40, 44, 45, 62– 65, 68– 70, 88, 91). It was established that this bacterium possessed the novel ability to degrade a variety of aromatic compounds including toluene, all isomers of xylene, p-cresol, naphthalene, biphenyl, dibenzothiophene, fluorene, salicylate, and benzoate ( 20, 22). Sphingomonas aromaticivorans F199 was isolated from sediments collected 410 m below the land surface near Allendale, S.C., in 1988 ( 4, 21). Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes. was demonstrated, and genes associated with this function were found in two large clusters. ![]() Conjugative transfer of pNL1 to another Sphingomonas sp. ![]() Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined.
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