Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-06-03
1999-02-16
Arthur, Lisa B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, 4353201, 536 237, 530350, 935 77, 935 78, C12Q 168, C07H 2104, C12P 1934, C07K 1400
Patent
active
058719229
DESCRIPTION:
BRIEF SUMMARY
This application was filed under 35 U.S.C. 371 from PCT/GB95/01125 filed May 18, 1995.
FIELD OF THE INVENTION
The present invention relates to the bacterial gene expression of carbapenem antibiotics.
BACKGROUND OF THE INVENTION
The carbapenem antibiotics constitute a diverse group of .beta.-lactam antibiotics characterised by potent anti-bacterial and .beta.-lactamase-resistant activity. More than forty different carbapenems are known, most of which are produced by the actinomycetes, particularly Streptomyces spp (Ratcliffe and Albers-Schonberg, 1982; Brown 1984; Williamson 1986; all cited in Baiton et al 1992).
Carbapenems have been isolated from the Gram-negative bacterium Serratia marcescens and Erwinia carotovora by Parker et al. (1982) and in Azospirillum spp UK 1521 by Kintaka et al. (1985); all cited in Bainton et al 1992.
Bainton et al (1992) have recently shown that carbapenem biosynthesis is regulated by the regulatory factor N-(3-oxohexanoyl)-L-homoserine lactone (known as HSL or OHHL). This compound was previously only known for its role in auto-induction of bioluminescence in the marine bacterium Vibrio fischeri. OHHL is also structurally related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the cosmid cWU142 and the organization of the car genes R,A,B,C,D,E,F,G,H.
FIG. 2 shows plasmid pSMG12.
FIG. 3 shows plasmid pSMG13.
FIG. 4 shows the entire nucleotide and predicted amino acid sequence of carR, carA, carB, carD, carE, carF, carG and carH.
DETAILED DESCRIPTION OF THE INVENTION
In order to examine the biosynthetic and regulatory mechanism involved in the production of the .beta.-lactam antibiotic 1-carbapen-2-em-3-carboxylic acid by Erwinia carotovora, blocked mutants were obtained with a carbapenem non-producing phenotype (Car ) as described by Bainton et al (1992). These mutants fell into two distinct groups: group 1 mutants secreted a low molecular mass diffusible factor which restored carbapenem biosynthesis in group 2 mutants, but not vice versa. This factor was shown to be OHHL. Class 1 mutants produced OHHL and were thus thought to be defective in carbapenem biosynthetic genes.
In order to study class 1 mutants a chromosomal DNA cosmid library of Erwinia carotovora strain ATCC 39048 was constructed in Escherichia coli. The cosmids produced were used in standard complementation studies to find a sequence which could restore the carbapenem antibiotic production in the class 1 mutants.
One cosmid (cWU142) was presumed to contain the carbapenem biosynthetic genes. Restriction fragments of this cosmid were sub-cloned and a 3.8 Kb EcoRI fragment was found to complement 7 out of 8 class 1 mutants. This fragment was sequenced and shown to comprise 2 Kb of cosmid DNA and 1.8 Kb of Erwinia DNA. This was extremely unexpected because known antibiotic biosynthetic gene sequences are much longer than 1.8 Kb.
The 1.8 Kb gene sequence was found to encode CarR, a homologue of the LuxR regulatory protein from the Lux operon system associated-with the bioluminescence phenotype of the marine bacterium V. fischeri.
In V. fischeri OHHL synthesis is coded for by the gene luxI. In Erwinia carotovora, OHHL synthesis is encoded by a luxI homologue which has been named carI.
By analogy with the V. fischeri Lux system, the inventors postulated that when OHHL is made, it binds to CarR which can then act as a transcriptional activator of the carbapenem biosynthetic genes. Thus, the inventors reasoned that 7 out of 8 of the class 1 mutants are not, as expected, defective in genes required for synthesis of carbapenem, but in a gene encoding a regulatory protein CarR, needed to switch on the carbapenem biosynthesis genes. Without the carR gene product the carbapenem biosynthetic genes are not expressed, that is, they remain silent or cryptic.
Our co-pending application GB-9311641.6 relates to the carR gene product and to DNA sequences encoding it.
We therefore used direct cosmid complementation of
REFERENCES:
Hase et al. Biochim. Biophys. ACTA 744(1):46-52, 1983.
Swift et al. Molecular Microbiology 10(3):511-520, 1993.
McGowan et al. Molecular Microbiology 22(3):415-426, 1996.
Keller et al. DNA Probes. M Stockton Press, New York, N.Y., pp. 255-259, 325-337, 1993.
Bycroft Barrie Walsham
Cox Anthony Richard John
Holden Matthew Thomas Geoffrey
McGowan Simon James
Porter Lauren Elizabeth
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