Conjugation and Plasmid R1
Bacterial Conjugation
Bacterial conjugation is one of the major processes by which genetic material
is transferred between bacteria. This type of horizontal gene transfer
provides the basis for the rapid spread of antibiotic resistance genes
among bacterial populations. The importance of gene transmission within
the microbial population is becoming increasingly apparent as multidrug-resistent
bacteria are emerging at an alarming rate. Plasmid R1 is a conjugative
resistance plasmid belonging to the incompatibility class IncFII. Genes
required for conjugation are organized in the so called resistance transfer
region. This region is very similar to the transfer region of the related
F-plasmid for which complete sequence information is available.
Based on their functions, the transfer genes can be classified as genes
required for i) pilus synthesis and assembly, ii) control of transfer gene
expression, iii) surface exclusion, iv) aggregate stability, and v) signal,
origin nicking, unwinding and transport.
Extensive sequence comparisons suggest relationships between F transfer
proteins and proteins from other DNA transfer systems like the Agrobacterium
tumefaciens Ti system.
Plasmid R1
Our studies focus on a DNA fragment from plasmid R1 which contains the
origin of transfer (oriT), that is the region where one strand of
DNA is cleaved by an endonuclease in preparation for DNA transfer from
a donor to a recipient cell. At the Institute of Microbiology in Graz we
sequenced this DNA fragment and identified nine genes surrounding the origin
of transfer. Four of these genes, traY, traA, traL, and traE
(tra: DNA transfer), constitute the beginning of the tra-operon,
a set of at least 34 genes, necessary for conjugation. The figure shows
the organization of genes around oriT. 
Genes of Interest
Plasmid R1 belongs to the so called F-like plasmids, relatives of the prototypic
fertility factor, F. It is, in the wild-type form, repressed, i.e. only
one out of ca. 1000 plasmid-carrying cells is able to participate in conjugation.
We established that an antisense RNA, called FinP
(fin: fertility inhibition), in conjunction with the protein FinO, constitutes
a repressor for conjugative DNA transfer.
The second type of RNA we are interested in is the mRNA of the
traA gene. It is the only stable segment
of the polycistronic tra-operon mRNA and its enhanced stability
enables the cell to produce large amounts of the TraA protein.
FinP antisense RNA and traA mRNA can be regarded as regulatory elements
of the plasmid. Further work in our laboratory revealed, that post-transcriptional
control is involved in the expression of another transfer function. The
expression of gene 19 , a gene which is
proximal to the origin of transfer, is controlled by the cleavage action
of RNase III on a hairpin structure within gene 19 mRNA. Our work
also showed that gene 19 is a very important gene in bacterial conjugation
and RNA phage infection. Its function is not
yet completely understood, but amino acid sequence comparisons suggest
that it encodes a transglycosylase. Transglycosylases are assumed to break
down the peptidoglycan layer at certain sites and thereby form holes that
would, in the case of gene 19 , be used for the DNA and phage RNA
transfer. Control of the amount of mRNA of this gene through RNase III
cleavage might help to limit expression of the gene to the physiologically
correct time.
Another gene that we have studied is gene traM
. We have shown that TraM is a DNA-binding protein. It binds to two
closely spaced regions upstream of gene traM resulting in autoregulation
of expression. We demonstrated that TraM binding to the DNA is mediated
by an amphiphilic N-terminal a-helix. The significance of DNA-binding of
TraM for conjugation is still not clear but mutational analysis in our
laboratory suggests that TraM binding upstream of gene traM is important
for regulation of tra-operon expression.
Recently, an assay could be developed which measures the site-
and strand-specific cleavage at the origin of DNA transfer, oriT,
that occurs as a first step in plasmid transfer. Surprisingly, we found
that some DNA is nicked in the absence of recipients, meaning that the
cleavage reaction is also occurring in the donor cells without the involvement
of the recipients.
Selected Publications
Koraimann, G., and G. Högenauer (1989) A stable core region of the
tra operon mRNA of plasmid R1-19. Nucl. Acids Res. 17: 1283-1297.
Koraimann, G., Koraimann, C., Koronakis, V., Schlager, S., and
G. Högenauer (1991) Repression and derepression of conjugation of
plasmid R1 by wild-type and mutated finP antisense RNA. Mol. Microbiol.
5: 77-87.
Schwab, M., Reisenzein, H., and G. Högenauer (1993) TraM
of plasmid R1 regulates its own expression. Mol. Microbiol. 7: 795-803.
Koraimann, G., Schroller, C., Graus, H., Angerer, D., Teferle,
K., and G. Högenauer (1993) Expression of gene 19 of the conjugative
plasmid R1 is controlled by RNase III. Mol. Microbiol. 9: 717-727.
Bayer, M., Eferl, R., Zellnig, G., Teferle, K., Dijkstra, A.,
Koraimann, G., and G. Högenauer (1995) Gene 19 of plasmid R1 is required
both for efficient conjugative DNA transfer and bacteriophage R17 infection.
manuscript submitted
Grants
Grants from the Austrian Science Foundation (FWF):
Genetics of the resistance plasmid R1: P5634, P6254, P7552, P9141
Participants in the Project
Doris Angerer- Michaela Bayer- Robert Eferl- Rainer Fratte- Hans Graus-
Annemarie Graus-Göldner- Harald Gruber- Bettina Jauk- Claudia Koraimann-
Günther Koraimann- Vassilis Koronakis- Gertrude Markolin- Regina Mitteregger-
Andrea Pichler- Hermann Prüger- Edith Rassi- Helga Reisenzein- Wilfried
Renner- Karin Teferle- Sabine Schlager- Christa Schroller- Margit Schwab-
Siegfried Wagner- Manuel Wenieris- Wolfgang Woger- Silvia Woschitz- Ellen
Zechner