Studies on actinomycete plasmid and bacteriophage DNA
Date
2011-07-07
Authors
Shibayama, Youtaro
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Abstract
The actinomycetes are a diverse group of Gram-positive, high G+C content bacteria of
the order Actinomycetales. Many species of this group are of human interest as a result of
their pathogenic nature and diverse metabolic properties. Those from the genus
ocardia
cause opportunistic infections of lung, brain and central nervous system, and cutaneous
tissue. They are also producers of antibiotics and industrially important enzymes. As
studies describing plasmids in this genus are limited, we have characterized a 4326 bp
cryptic plasmid pYS1 from
ocardia aobensis IFM 10795. Three open reading frames
(ORFs) were predicted. Both sequence analyses and detection of single-stranded
intermediates suggested a rolling-circle mechanism as the mode of replication of pYS1.
Mutageneses and deletion analyses revealed both the predicted double- and singlestranded
origins to be indispensable in replication and thus lack of secondary signals for
both leading and lagging strand synthesis. The replicon of pYS1 is broad-host-range and
compatible to that of pAL5000 of mycobacteria, making it potentially useful in genetic
manipulation of various actinomycetes. Insertion analyses showed orf1, despite its
sequence similarity to plasmid transfer genes, is involved in plasmid stability rather than
conjugation and is lethal in the absence of a functional orf3. This situation is somewhat
analogous to the kil/kor system of pIJ101 of Streptomyces, except that orf3 was unrelated
to korA and was shown by promoter-probe assays to encode a novel transcriptional
repressor negatively regulating orf1 expression.
Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for 8 to
10 million new cases of disease and 3 million deaths every year, many of which in
HIV/AIDS patients. To identify novel drug targets in multi-drug resistant strains, DNA
from Rhodococcus phages were tested and shown to be inhibitory to mycobacteria. To
study the modes of action of products encodes by these genes, the lambda cI857 repressor
and PR promoter system was tested for its ability to function as a tight genetic switch for
toxic gene expression. Induction studies in both E. coli and mycobacteria indicate that
strong mycobacterial promoters may be necessary to drive expression of toxic genes and/or the repressor gene.