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Belas R, Bartlett D, Silverman M. Cloning and Gene Replacement Mutagenesis of a Pseudomonas atlantica Agarase Gene. Appl Environ Microbiol. 1988;54(1):30-37doi: 10.1128/aem.54.1.30-37.1988.
Belas, R., Bartlett, D., & Silverman, M. (1988). Cloning and Gene Replacement Mutagenesis of a Pseudomonas atlantica Agarase Gene. Applied and environmental microbiology, 54(1), 30-37. https://doi.org/10.1128/aem.54.1.30-37.1988
Belas, Robert, et al. "Cloning and Gene Replacement Mutagenesis of a Pseudomonas atlantica Agarase Gene." Applied and environmental microbiology vol. 54,1 (1988): 30-37. doi: https://doi.org/10.1128/aem.54.1.30-37.1988
Belas R, Bartlett D, Silverman M. Cloning and Gene Replacement Mutagenesis of a Pseudomonas atlantica Agarase Gene. Appl Environ Microbiol. 1988 Jan;54(1):30-37. doi: 10.1128/aem.54.1.30-37.1988. PMID: 16347536; PMCID: PMC202392.
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Appl Environ Microbiol. 1988 Jan;54(1):30-37. doi: 10.1128/aem.54.1.30-37.1988.

Cloning and Gene Replacement Mutagenesis of a Pseudomonas atlantica Agarase Gene.

Applied and environmental microbiology

Robert Belas, Douglas Bartlett, Michael Silverman

Affiliations

  1. Agouron Institute, La Jolla, California 92037.

PMID: 16347536 PMCID: PMC202392 DOI: 10.1128/aem.54.1.30-37.1988

Abstract

An agarase gene (agrA) was isolated by cloning genomic DNA prepared from Pseudomonas atlantica. The agarase activity in recombinant Escherichia coli was found in cell-free culture supernatants and could pass through a 0.45-mum-pore-size membrane separating cells from agar, suggesting that the gene product was exported in E. coli. The enzyme was specific for agar and agarose and did not digest alginate or carrageenan. Mutations generated by transposon mini-Mu d1(lacZ Km) were used to define the agrA coding region, as well as the direction of transcription of the gene. A procedure was developed to produce a P. atlantica agrA mutant. This required construction of an agrA::kan insertion mutation in vitro and subsequent introduction of the defect into the chromosome of P. atlantica by recombinational exchange. Transformation of P. atlantica with plasmids containing agrA::kan utilized a Tris-polyethylene glycol 6000-CaCl(2) treatment for making competent cells. Replacement of wild-type agrA with agrA::kan resulted in loss of agarase activity. Uses of the agrA gene probe and an Agr mutant for environmental studies are discussed.

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