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Honours Projects for 2009 in the Molecular
Genetics Laboratory
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The Molecular
Genetics Lab is an ideal
environment to undertake Honours studies, providing state-of-the-art-facilities with support from an active team of
postdoctoral researchers, and PhD and other Honours
students. Specific Honours projects are described below, and if you are
interested in doing Honours with us, please contact us to come in for a chat,
meet the crew and tour the facilities. Further
information about Honours in the School of Biological Sciences is available here.
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Partitioning Systems of Staphylococcal Multiresistance Plasmids |
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The S. aureus plasmids pSK1 and pSK41 are prototypes of two families of clinically important staphylococcal multiresistance plasmids. These plasmids possess two distinct plasmid maintenance determinants that contribute to their segregational stability in bacterial populations, even in the absence of selection for the resistance phenotypes that they confer. pSK41 encodes a resolvase, which converts plasmid multimers into monomers, and a type II partitioning system which moves plasmid copies into daughter cells during cell division. This par system encodes a DNA binding protein, ParR, a filament forming actin homologue, ParM, and a cis-acting centromere-like DNA site that ParR binds to. pSK1 also encodes a resolvase, but does not possess a typical partitioning system. Instead, a single protein-encoding gene, now designated par, located immediately upstream of, and divergently transcribed from, the rep gene, has been shown to enhance plasmid maintenance; this protein has been shown to bind to a DNA site just upstream of its coding sequence.
Honours
projects will focus on the protein-DNA interactions to identify
critical amino acids in the proteins and nucleotides in their cognate
DNA binding sites. Additionally protein-protein interactions can be
investigated. Methods will include site-directed mutagenesis in
combination with segregational stability assays to assess the impact of
mutations in vivo. Effects on DNA binding will be evaluated in vitro
using electrophoretic mobility shift assays and DNase I footprinting,
facilitated by protein overexpression and purification. Both proteins
have autoregulatory roles so the impact of mutations on transcription
of their respective promoters will be examined using reporter gene
fusions. Cytological studies such as immuno-fluorescence microscopy
(IFM) and fluorescence in situ hybridisation (FISH) might also be used
to investigate the cellular localisation of the proteins. Relevant
Publications Berg, T., N. Firth, S, Apisiridej, A. Hettiaratchi, A. Leelaporn and R.A. Skurray (1998). J. Bacteriol. 180:4350-4359. Schumacher, M.A., T.C. Glover, A.J. Brzoska, S.O. Jensen, T.D. Dunham, R.A. Skurray and N. Firth (2007). Nature 450:1268-1271.
Firth, N., S. Apisiridej, T. Berg, B.A. O’Rourke, S. Curnock, K.G.H. Dyke and R.A. Skurray (2000). J. Bacteriol. 182: 2170-2178. |
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Coordinate Regulation of a Conjugative
Staphylococcal Multiresistance Plasmid by a Global Regulator of Transcription
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In staphylococci, antimicrobial resistance determinants are often found to reside on plasmids. Several mechanisms of genetic exchange facilitate the transfer of plasmids, and hence resistance genes, between cells. The presence of plasmids thus accelerates the acquisition and spread of antimicrobial resistance in the hospital environment. The conjugative multiresistance
plasmid pSK41 confers resistance to the aminoglycoside antibiotics
gentamicin, tobramycin, kanamycin and neomycin, as well as multidrug
resistance to antiseptics and disinfectants. It is the prototype of a family
of clinically important plasmids, which includes members that also encode
resistance to vancomycin, trimethoprim, penicillin and mupirocin. Most of the
genes involved in conjugative transfer of pSK41 are organised into two
operons within the transfer (tra) region. One gene located in the tra region, artA, is divergently
transcribed from the two tra operons and encodes a repressor protein that
regulates transcription of all three tra
region promoters. These promoters all contain repeated copies of the
sequence 5'-CATGACA-3' overlapping their –35 regions which is
predicted to constitute the operator sequence recognised by ArtA.
Analysis of the entire 46 kb pSK41 nucleotide sequence has revealed an
additional seven promoters with CATGACA-like sequences. As four of the
ten promoters potentially regulated by ArtA are likely to direct
transcription of operons, it is conceivable that ArtA regulates
expression of 24 of the 29 coding sequences contained in the pSK41
backbone. In addition to regulating transfer genes, ArtA might also
modulate transcription of genes involved in plasmid maintenance.
Therefore, ArtA may be a global regulator that coordinates the
expression of housekeeping functions of pSK41 family plasmids, allowing
them to confer multiple resistance phenotypes without imposing undue
energetic burden on their hosts. The
Honours project will focus on the interaction between the ArtA protein
and its DNA operator sites. Specific questions to be addressed are
which parts of the protein are involved in DNA binding and
protein-protein interactions, and which nucleotides in the operator
sequences are critical to for ArtA binding. Methods will include
site-directed mutagenesis in combination with assays using
promoter-reporter gene fusions to assess the impact of mutations in
vivo. Effects on DNA binding at operators will be evaluated in vitro
using electrophoretic mobility shift assays and DNase I footprinting,
facilitated by ArtA overexpression and purification. Relevant
Publications Firth, N. and R.A. Skurray (2006). In, Gram-Positive Pathogens, 2nd Edition. ASM Press. Washington D.C. p.413-426. |
Transcriptional organization of conjugative multiresistance plasmid pSK41 |
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Molecular Analysis of Staphylococcal
Multiresistance Plasmid Replication |
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In staphylococci, antimicrobial resistance determinants are often found to reside on plasmids. Several mechanisms of genetic exchange facilitate the transfer of plasmids, and hence resistance genes, between cells. The presence of plasmids thus accelerates the acquisition and spread of antimicrobial resistance in the hospital environment. Several classes of plasmids have been identified in S. aureus and they range from small plasmids that replicate by a rolling-circle mechanism and may be cryptic or encode a single resistance determinant to large plasmids (>40 kb) that carry multiple resistance determinants. The multiresistance plasmids are thought to replicate through a theta-like mechanism and are broadly divided into three categories; namely, the beta‑lactamase/heavy-metal resistance plasmids, the pSK1 family and the conjugative pSK41-like plasmids. The predicted replication initiation proteins from representatives of all three categories (pI9789, pSK1 and pSK41, respectively) were found to share considerable amino acid sequence homology, establishing that all three recognized groups of large staphylococcal multiresistance plasmids utilise evolutionarily related theta-mode replication systems. This single type of replication system has therefore had a major impact on the worldwide development of antimicrobial resistant staphylococci. However, relatively little is known at the molecular level about the replication mechanism(s) of these staphylococcal multiresistance plasmids. The conjugative multiresistance
plasmid pSK41 (46.4-kb) confers resistance to the aminoglycoside antibiotics
gentamicin, tobramycin, kanamycin and neomycin, as well as multidrug
resistance to antiseptics and disinfectants. Recently, we have over-expressed
and purified a recombinant form of the pSK41 replication initiation protein,
Rep. Using electrophoretic mobility shift assays and DNase I footprinting we
have shown that the Rep protein binds in vitro to DNA sequences located
centrally within the pSK41 rep coding region. The binding-site
contains four tandemly repeated sequences that are predicted to be essential
for pSK41 origin of replication (oriV) activity. The aim of this
project is to elucidate the molecular basis of the interaction between the
pSK41 Rep protein and oriV. Random and site-directed PCR-based mutagenesis of
the rep gene will be undertaken to identify, firstly, amino acids within Rep
required for DNA binding, and secondly, residues required for replication but
not involved in DNA binding. Mutant Rep proteins will be over-expressed and
purified by affinity chromatography and their DNA-binding ability determined
using electrophoretic mobility shift assays. The activity of the mutant
proteins in vivo will be determined using plasmid replication assays.
Additionally, mutagenesis of the sequence repeats within oriV will be undertaken to
demonstrate their functional significance. The project provides an
opportunity to learn a wide range of molecular biology/genetics techniques,
and there is scope to incorporate bioinformatic analyses for students with
skills in this area. Several other honours projects that investigate the
replication and maintenance of staphylococcal multiresistance plasmids will
also be available for discussion with interested students.
Firth, N. and R.A. Skurray (2006). In, Gram-Positive Pathogens, 2nd Edition. ASM Press. Washington D.C. p.413-426. Kwong, S.M. and N. Firth (2006). Regulatory RNA molecules. Microbiol. Aust. 27: 124-127. Kwong, S.M., R.A. Skurray and N. Firth (2006). J. Bacteriol. 188: 4404-4412.
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The pSK41 replication region
Structure predications of pSK41 replication region RNA transcripts |
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What turns a commensal bacterium Staphylococcus epidermidis into a human pathogen? |
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Staphylococcus epidermidis is the most common staphylococcal species found on human skin. It is an opportunistic pathogen, which because of its ability to form biofilms, is a particular problem for patients with implanted medical devices. Non-aureus staphylococci, particularly S. epidermidis, are among the five most commonly reported pathogens in hospitals and the most frequently reported isolates in nosocomial bloodstream infections. The most critical step in the pathogenesis of S. epidermidis foreign body-associated infections is the colonisation of the polymer surface by the formation of multilayered cell clusters within a biofilm of bacterial and host extracellular products. Here the bacteria can survive and proliferate with reported resistance to host defence systems. Subsequent dissemination from the infected medical device can lead to circulation in the host organism, leading to further stimulation of inflammatory host responses and potential colonisation of other infection sites. S. epidermidis isolates in the hospital environment have been shown to differ from those obtained outside of medical facilities with respect to biofilm formation, antibiotic resistance and the presence of mobile DNA elements. The genetic and selective drivers behind the transition of strains of S. epidermidis from human commensal to pathogen are poorly understood and certainly not studied in the Australian context. Nine complete genomes of S. aureus, and two genomes of S. epidermidis,
one of a pathogenic isolate, and the other non-pathogenic, are publicly
available. A comparative bioinformatic analysis of these staphylococcal
genomes that we conducted earlier this year identified a number of
unique genes for virulence and resistance that are candidates for
further study into the evolution of pathogenic and commensal forms of
staphylococci. |
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How are the genetic determinants for antimicrobial resistance exchanged amongst pathogenic staphylococci? |
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Genetic exchange is believed to play a key role in the evolution of human pathogenic bacteria with resistance to multiple antimicrobial agents. It is well established that staphylococci, like most bacteria, are able to exchange genetic determinants for potentially useful characters, including antimicrobial resistance, horizontally between strains, species and even genera. The evidence is implicit in the possession of identical plasmids and mobile genetic elements (transposons) by otherwise unrelated strains, while specific gene transfer has been demonstrated in a limited number of laboratory experiments. Members of the family of large staphylococcal multiresistance plasmids of which pSK41 is the prototype have been shown to encode their own transfer through a conjugative mechanism analogous to that seen in Gram-negative organisms such as E. coli. The existence of these conjugative plasmids can explain the dissemination of a few, but not all, genetic determinants of antimicrobial resistance found within populations of staphylococci. Staphylococci also display a range of smaller non-conjugative multiresistance plasmids such as pSK1, which have been observed to transfer to recipient cells in the absence of identifiable transfer (tra) genes. It
is unlikely that naked DNA transformation is effective in natural
populations of staphylococci due to nucleases secreted into the
environment by these species. This then leaves some form of genetic
transfer mediated by bacteriophages as the only likely process for the
past and present exchange of the majority of staphylococcal
antimicrobial resistance determinants. Conventional phage-mediated gene
transfer (transduction) has been shown to play a viable but perhaps
limited role in vivo. A
second process branded phage-mediated conjugation (also known as
mixed-culture transfer) stands out as a potentially huge contributor to
genetic exchange, due to its high frequency and ability to transfer
both plasmid and chromosomal genes. Surprisingly, little is known about
the molecular mechanism underlying mixed-culture transfer, apart from a
requirement for the presence of a prophage in either donor or recipient
together with high cell density and calcium ions. |
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(c)
The University of Sydney, NSW 2006 Australia. |
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ABN:
15 211 513 464 CRICOS Number: 00026A |
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Authorised
by: School of Biological Sciences. Last Updated: 28/5/08 by Neville Firth |