Manasiev Jordan, Gerginova Maria, Yemendzhiev Hyusein, Peneva Nadejda, Alexieva Zlatka
Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bontchev Str., bl. 26, 1113 Sofia, Bulgaria.
Z Naturforsch C J Biosci. 2008 Jan-Feb;63(1-2):133-8. doi: 10.1515/znc-2008-1-224.
In an attempt to estimate the occurrence of phenol hydroxylase-related gene sequences we performed a dot blot hybridization assay with DNA from phenol utilizing Trichosporon cutaneum R57 strain NBIMCC 2414 and microbial isolates from different wastewaters. The used oligonucletides were homologous to the 5'-end of TORPHD locus (NCBI)-coding phenol hydroxylase in Trichosporon cutaneum ATCC 46490 and to the 5'-end of TORCCMLE locus (NCBI)-coding cis,cis-muconate-lactonizing enzyme in Trichosporon cutaneum ATCC 58094. Two microbial strains, Escherichia coli JM 109 and Lactobacillus acidophilus ATCC 4356, incapable to degrade phenol were used as negative controls. We established the presence of hybridization with both used oligonucleotide probes in T. cutaneum R57 and T. cutaneum ATCC 46490 yeast strains. The experiments implemented with microbial isolates obtained from three industrialized areas in Bulgaria showed that 7 of them may carry sequences hybridizing with a phenol hydroxylase oligonucleotide probe. A subsequent hybridization test for the cis,cis-muconate-lactonizing enzyme showed that only 3 of them displayed a positive signal. Lactobacillus acidophilus ATCC 4356 and Escherichia coli JM 109 strains' DNA used as negative controls in the experiments did not reveal any sequence similarity to the both applied oligonucleotides. The partial nucleotide sequences of 16S rDNAs of the isolated strains C1 and K1 obtained as PCR products were determined and sequenced. A comparison of these nucleotide sequences with similar sequences in NCBI Data Bank indicated that both C1 and K1 strains are closely related to the genera Acinetobacter and Burkholderia.
为了估计苯酚羟化酶相关基因序列的出现情况，我们利用皮状丝孢酵母R57菌株NBIMCC 2414和来自不同废水的微生物分离株的DNA进行了斑点杂交试验。所用的寡核苷酸与皮状丝孢酵母ATCC 46490中编码苯酚羟化酶的TORPHD基因座（NCBI）的5'端以及皮状丝孢酵母ATCC 58094中编码顺，顺-粘康酸内酯化酶的TORCCMLE基因座（NCBI）的5'端同源。两种不能降解苯酚的微生物菌株，大肠杆菌JM 109和嗜酸乳杆菌ATCC 4356，用作阴性对照。我们确定在皮状丝孢酵母R57和皮状丝孢酵母ATCC 46490酵母菌株中，两种所用的寡核苷酸探针均存在杂交现象。对从保加利亚三个工业化地区获得的微生物分离株进行的实验表明，其中7个可能携带与苯酚羟化酶寡核苷酸探针杂交的序列。随后对顺，顺-粘康酸内酯化酶进行的杂交试验表明，其中只有3个显示出阳性信号。实验中用作阴性对照的嗜酸乳杆菌ATCC 4356和大肠杆菌JM 109菌株的DNA未显示与两种应用的寡核苷酸有任何序列相似性。对作为PCR产物获得的分离菌株C1和K1的16S rDNA的部分核苷酸序列进行了测定和测序。将这些核苷酸序列与NCBI数据库中的相似序列进行比较表明，C1和K1菌株均与不动杆菌属和伯克霍尔德菌属密切相关。
