5 to 99. 3% sequence identity together with the PhnAc sequences iden tified to date from pure cultures. Phylogenetic examination of PhnAc like sequences retrieved from sediment libraries as well as the ones from pure cultures uncovered two clades, one particular containing phenanthrene dioxygenase sequences in the isolates Burkholderia sp. strains Cs1 four, Ch1 1, Ch3 five and Eh1 1 as well as a second one with PhnAc from A. faecalis AFK2, Representatives from both clades had been identified from the Ac OR04 library. Alternatively, all Ac SC04 and Ac MS05 PhnAc like sequences clustered inside of the Burkholderia clade, when all Ac GR06 PhnAc like gene fragments clustered inside of the A. faecalis AFK2 clade, NahAc style sequences formed two distinct, extremely sup ported groups, 1 of them incorporated sequences from Pseudomonas stutzeri strain AN10, Pseudomonas aeruginosa PAK1 and Pseudomonas balearica SP1402 plus the other clade incorporated sequences belonging to P.
selleckchem putida strains NCIB 9816 4, G7, OUS82 and other connected Pseudomonas isolates, The two groups incorporate sequences from marine isolates and also have been described previously as AN10 and C18 groups respectively, Only sequences from the C18 cluster were detected in coastal sediments. This isn’t sudden since the AN10 group was most almost certainly not targeted from the primers utilised in this work, Construction and examination of phnA1 like ARHD gene libraries A primer set was made to target phnA1 like ARHD sequences from bacteria belonging to the genus Cycloclas ticus. This primer set effectively amplified a 500 bp frag ment from Cycloclasticus pugetii PS 1, which was confirmed like a phnA1 gene fragment by sequencing.
Amplifications with this primer set also resulted in solutions in the anticipated dimension in samples MP04, GR06, selleck chemicalWZ4003 AR06, SC04, OR04 and OR05, Samples PF05, MS05, CR05, OR06, EM06 and OL06 weren’t tested with this particular primer set. The amplification items of samples SC04 and OR05 had been cloned and analyzed by RFLP examination together with the RsaI restriction endonuclease. As each of the analyzed clones showed restriction patterns identi cal for the 1 produced from the amplification products of C.