coliDH5α, and plated onto LB agar supplemented with 100 μg of kanamycin per ml. Ligation mixtures were packaged into lambda particles by using the Gigapack II packaging extract (Stratagene, La Jolla, Calif.), infected into E. For construction of the large insert library, a partial Sau3A digestion of pNL1 was used to produce fragments of approximately 40 kb, which were then dephosphorylated and ligated into the BamHI site of the sCos-1 cosmid vector ( 16). The procedure for isolation of pNL1 has been described elsewhere ( 86). Conditions for testing the ability to grow on aromatic compounds or to produce colored intermediates from them by Sphingomonas strains have been described elsewhere ( 20). The resulting Sphingomonas insertion mutants were screened for loss of dioxygenase activity by placing a small crystal of indole in the lid of the petri dish the colonies were then examined for the absence of blue coloring (i.e., lack of indigo formation). coli colonies because of their characteristic yellow pigmentation. Sphingomonas colonies are readily distinguished from E. The resulting bacterial spots were resuspended in 1 ml of saline, and 100-μl aliquots were plated onto 0.5× LB containing kanamycin to select for transposition and polymyxin B to select against the E. coli with either SB354 or pRK2013, were then overlaid, dried, and incubated overnight at 30☌. An equivalent amount of overnight cultures of the donor strains, E. aromaticivorans F199 recipient was spotted and allowed to dry on half-strength LB agar. This unusual gene arrangement suggests that a highly complex regulatory network is responsible for the expression of aromatic degradative pathways in some Sphingomonas spp.Ī portion (50 μl) of an overnight culture of the S. 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 Sphingomonasbiodegradative 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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