| Non-Scientific Abstract It has been known for a long time that bacteria
have the ability to exchange genetic information between each other. Scientists did not begin to
understand the way in which this process occurs, however, until they discovered that bacteria have
small circular strands of genetic information called plasmids (in addition to their chromosomes).
Bacteria can produce copies of these plasmids via replication and then transfer the genetic
information to other bacteria through a process called conjugation.The plasmid contains genes that
encode proteins necessary for conjugation to occur, as well as other components such as the sequence
at which conjugation begins (known as the origin of transfer or oriT). The purpose of this study was
to identify specific regions of a plasmid called pB264, found in Rhodococcus sp. B264-1, that
are critical for its transfer to another strain of bacteria known as Rhodococcus sp. I24. By
taking out parts of the plasmid or adding in other sequences to the plasmid and then observing whether
or not the plasmid could still be transferred between the bacteria, it could be determined which
regions of the plasmid are necessary for conjugation to occur and which regions are not. Through this
study, we were able to locate a certain section of the plasmid that might be involved in conjugation
and should be further examined.
Abstract
Several bacteria belonging to the genus Rhodococcus have exhibited the ability to degrade a wide
range of xenobiotic compounds, as well as the ability to synthesize products useful to various
industries. It is thought that Rhodococci may have obtained the genes encoding such
capabilities from other genera of bacteria through horizontal gene transfer, possibly via conjugation.
In this project,we studied the conjugal transfer of a 4851-bp plasmid pB264 from Rhodococcus
sp. B264-1 to Rhodococcus sp. I24 in an attempt to understand the possible mechanism of
conjugation among these bacteria.Using an 8642-bp plasmid, pJANET, that contains pB264 ligated to an
Escherichia coli vector, we performed filter mating experiments and confirmed that conjugation
occurs between these two bacterial strains. Through transposon mutagenesis, deletion analysis, and
reconstruction analysis, we created plasmids with modifications of pB264 that will be useful in
identifying its critical transfer regions. Two Rhodococcus sp. B264-1 mutants carrying putative
transposon insertions in pB264 have been isolated. In addition,we successfully constructed four pJANET
plasmids with distinct deletions in the pB264 element and cloned 1-kb segments of pB264 into
pCR2.1-TOPO. Examination of the pB264 sequence for inverted repeats suggested that the region around
nucleotides 4014-4035 might play a role in conjugal transfer and should be more closely investigated.
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