Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains

被引:166
作者
Smits, THM [1 ]
Röthlisberger, M [1 ]
Witholt, B [1 ]
van Beilen, JB [1 ]
机构
[1] ETH Honggerberg, Inst Biotechnol, CH-8093 Zurich, Switzerland
关键词
D O I
10.1046/j.1462-2920.1999.00037.x
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C-6-C-11) Or long-chain n-alkanes (C-12-C-16), PCR products Of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2-93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxyiase. Strains that were unable to grow on n-alkanes did not yield non products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB, respectively, to grow on n-alkanes, showing that the cloned genes do indeed encode alkane hydroxylases.
引用
收藏
页码:307 / 317
页数:11
相关论文
共 60 条
[1]   BASIC LOCAL ALIGNMENT SEARCH TOOL [J].
ALTSCHUL, SF ;
GISH, W ;
MILLER, W ;
MYERS, EW ;
LIPMAN, DJ .
JOURNAL OF MOLECULAR BIOLOGY, 1990, 215 (03) :403-410
[2]   SPECIFIC-PURPOSE PLASMID CLONING VECTORS .2. BROAD HOST RANGE, HIGH COPY NUMBER, RSF1010-DERIVED VECTORS, AND A HOST-VECTOR SYSTEM FOR GENE CLONING IN PSEUDOMONAS [J].
BAGDASARIAN, M ;
LURZ, R ;
RUCKERT, B ;
FRANKLIN, FCH ;
BAGDASARIAN, MM ;
FREY, J ;
TIMMIS, KN .
GENE, 1981, 16 (1-3) :237-247
[3]  
BIRNBOIM HC, 1979, NUCLEIC ACIDS RES, V7, P1513
[4]   CONSTRUCTION AND CHARACTERIZATION OF NEW CLONING VEHICLES .2. MULTIPURPOSE CLONING SYSTEM [J].
BOLIVAR, F ;
RODRIGUEZ, RL ;
GREENE, PJ ;
BETLACH, MC ;
HEYNEKER, HL ;
BOYER, HW ;
CROSA, JH ;
FALKOW, S .
GENE, 1977, 2 (02) :95-113
[5]  
CARDINI G, 1968, J BIOL CHEM, V243, P6070
[6]  
CLIFFORD KH, 1991, Patent No. 5037759
[7]   ILLEGITIMATE INTEGRATION OF NON-REPLICATIVE VECTORS IN THE GENOME OF RHODOCOCCUS-FASCIANS UPON ELECTROTRANSFORMATION AS AN INSERTIONAL MUTAGENESIS SYSTEM [J].
DESOMER, J ;
CRESPI, M ;
VANMONTAGU, M .
MOLECULAR MICROBIOLOGY, 1991, 5 (09) :2115-2124
[8]   PROPERTIES OF 6 PESTICIDE DEGRADATION PLASMIDS ISOLATED FROM ALCALIGENES-PARADOXUS AND ALCALIGENES-EUTROPHUS [J].
DON, RH ;
PEMBERTON, JM .
JOURNAL OF BACTERIOLOGY, 1981, 145 (02) :681-686
[9]   HIGH-EFFICIENCY TRANSFORMATION OF ESCHERICHIA-COLI BY HIGH-VOLTAGE ELECTROPORATION [J].
DOWER, WJ ;
MILLER, JF ;
RAGSDALE, CW .
NUCLEIC ACIDS RESEARCH, 1988, 16 (13) :6127-6145
[10]  
EGGINK G, 1987, J BIOL CHEM, V262, P17712