Honours Projects Opportunities in the Molecular Genetics Lab
- Partitioning systems of staphylococcal multiresistance plasmids. (Neville Firth & Slade Jensen)
- Molecular analysis of staphylococcal multiresistance plasmid replication. (Neville Firth & Stephen Kwong )
- What turns the commensal bacterium Staphylococcus epidermidis inta a human pathogen? (Bruce Lyon & Neville Firth)
- How are the genetic determinants for antimicrobial resistance exchanged amongst pathogenic staphylococci? (Bruce Lyon & Neville Firth)
Partitioning Systems of Staphylococcal Multiresistance Plasmids
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.
- 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.
- Simpson, A.E., R.A. Skurray and N. Firth (2003). J. Bacteriol. 185: 2143–2152.
Molecular Analysis of Staphylococcal Multiresistance Plasmid Replication
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 pSK41 replication region|
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.
|Structure predications of pSK41 replication region RNA transcripts|
- Berg, T., N. Firth, S, Apisiridej, A. Hettiaratchi, A. Leelaporn and R.A. Skurray (1998). J. Bacteriol. 180:4350-4359.
- 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.
- Kwong, S.M., R.A. Skurray & Firth, N. (2004). Mol. Microbiol. 51:497-509.
- 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.
What turns a commensal bacterium Staphylococcus epidermidis into a human pathogen?
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.
Honours projects will use DNA analysis and sequencing to make molecular genetic and epidemiological comparisons between clinical and community-acquired isolates obtained from a variety of sources. In vitro models for microbial biofilm formation will be employed to evaluate different strains for their ability to adhere to synthetic substrates, while microbial cell viability will be assayed to quantify adherent and planktonic bacterial cells. Genes inferred by bioinformatic analysis to be associated with pathological characteristics might be subjected to gene knock-out to determine their possible involvement in the virulence of S. epidermidis.
How are the genetic determinants for antimicrobial resistance exchanged amongst pathogenic staphylococci?
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.
Honours projects will survey a range of clinical isolates of S. aureus and S. epidermidis for their ability to donate genetic determinants for antimicrobial resistance via mixed-culture transfer. Bioinformatic and molecular genetic techniques will then be employed to test the underlying basis for this phenomenon. For example, donor strains might be screened for the presence of prophage sequences, and such sequences could be knocked-out to observe any changes in transfer efficiency. Plasmids and mobile genetic elements could be similarly analysed for attributes that enable them to be efficiently exchanged via mixed-culture transfer.