Identification and expression of the cym, cmt, and tod catabolic genes from Pseudomonas putida KL47: expression of the regulatory todST genes as a factor for catabolic adaptation.
Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene (C(1)-C(4)), biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99%) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.
Lee
Kyoung
K
Department of Microbiology, Changwon National University, Kyongnam 641-773, Republic of Korea. kyounglee@changwon.ac.kr
Ryu
Eun Kyeong
EK
Choi
Kyung Soon
KS
Cho
Min Chul
MC
Jeong
Jae Jun
JJ
Choi
Eun Na
EN
Lee
Soo O
SO
Yoon
Do-Young
DY
Hwang
Ingyu
I
Kim
Chi-Kyung
CK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001565:Benzoates / Q000378:metabolism / Q000494:pharmacology; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D000081007:Cymenes; D015870:Gene Expression / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000378:metabolism / Q000494:pharmacology; D005810:Multigene Family / Q000235:genetics; D009154:Mutation; D009876:Operon / Q000235:genetics*; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D014050:Toluene / Q000378:metabolism / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D001565:Benzoates; D000081007:Cymenes; D039821:Monoterpenes; C104650:TodT protein, Pseudomonas putida; D015534:Trans-Activators; C007210:4-cymene; D014050:Toluene; C055607:cumic acid; D011494:Protein Kinases; C104649:TodS protein, Pseudomonas putida