Actinomycetes, 1996, Vol.7, Part 2. pp.55-65
THE CONSTRUCTION AND USE OF PROMOTER PROBE VECTORS
FOR RHODOCOCCUS SP.
K. J. KAYSER, C.-O. YUN and J. J. KILBANE II
Code Number: AC96009
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Text: 34.9K
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Abstract.
Three promoter probe vectors have been constructed for use
within Rhodococcus sp. They are hybrid replicons capable of
replicating both in E.coli and Rhodococcus species due to
the presence of replication functions derived from pUC19 and
the Rhodococcus fascians plasmid pRF29 respectively. Promoter
probe vector pRCM1 contains a promoterless gene which encodes
a membrane-associated chloramphenicol efflux protein (cmr)
derived from Rhodococcus fascians plasmid pRF2, pRCAT3
contains a promoterless chloramphenicol acetyl transferase
gene (cat) derived from Tn9, and pEBC26 contains a
promoterless beta-galactosidase gene derived from pSVB-gal.
Many derivatives of pRCM1 and pRCAT3 receiving inserts that
regulated the expression of chloramphenicol resistance in
Rhodococcus sp. strain IGTS8 proved to be unstable in E.coli,
frequently yielding plasmids containing deletions. This
instability was found to be largely associated with these
vectors; however, some inserts of Rhodococcus DNA increased
and others alleviated this instability. Derivatives of pEBC26
were stable both in Rhodococcus and E.coli and many DNA
fragments encoding Rhodococcus promoters were isolated. The
size of these promoter-containing DNA fragments ranged from
0.15 to 3 Kb and the level of beta-galactosidase expression in
Rhodococcus hosts ranged from 0.1 to 838 Miller units.
Promoters from Rhodococcus were not observed to function in
E.coli; however, the E.coli rrnB promoter was shown to
function weakly in Rhodococcus.
Copyright 1996 C.E.T.A., The International Centre for
Theoretical and Applied Ecology, Gorizia
