John Pemberton

My research interests have concentrated on the molecular genetic analysis of multigene phenotypes of bacteria encompassing pathogenicity, bacterial degradation of synthetic environmental pollutants, photosynthesis and the synthesis of antitumour antibiotics. My PhD research focussed on plasmids and mapping of the genome of the human pathogen P. aeruginosa (Pemberton,and Holloway, 1972a; Pemberton,and Holloway,1972b;Pemberton and Holloway,1973). I continued this research as a postdoc at UC Berkeley with John Clark in the Department of Molecular Biology in the Wendell Stanley Virus laboratory. I am grateful to Mark Guyer who taught me how to isolate large plasmid DNAs. In Robley Williams lab I learnt how to use the Kleinschmidt and Zahn technique for spreading the plasmid DNA on an electronmicroscope grid and metal shadow the sample to visualise it under an electron microscope; I am grateful to Robley Williams for showing me how to metal shadow my samples (Pemberton,1973; Pemberton and AJ Clark,1973; Miller, Pemberton and Richards,1974;Pemberton,1974;Miller,Pemberton and Clark,1977). After advice from John Clark and when I returned to Australia and took up an appointment with UQ I decided to diversify my research. During my postdoc I worked alongside Anne Emerick who was working with the CAM (camphor degradation) plasmid. John Clark put me on her advisory panel (alongside Mike Doudoroff and Norberto Palleroni) making her my first PhD student. The bacterial degradation of such complex naturally occurring molecules such as camphor required a large number of steps requiring a large number of genes hence a large plasmid. I decided to determine if soil bacteria had evolved plasmids which encoded the degradation of man-made molecules. I chose the synthetic herbicide 2,4-D. My research was the first to identify, isolate and clone genes responsible for the degradation of a man-made molecule –moreover the 2,4-D degradation was encoded by a broad host range plasmid, providing an explanation of how microorganisms rapidly evolve the ability to degrade and recycle a vast array of worldwide synthetic environmental pollutants which cause a range of diseases from cancer to birth defects (Pemberton & Fisher, Nature, 1977). One of the most widely studied microorganisms is the bacterium Ralstonia eutropha JMP134 pJP4 (Hgr) which has an extraordinary ability to degrade and recycle the most complex and most toxic synthetic molecules (Don and Pemberton, J.Bacteriol, 1981;Schmidt et.al.,2011. Catabolic Plasmids.Encyclopedia of Life Sciences). Famously more recent studies have shown that there are genes and gene clusters encoding the degradation of plastics, explosives and chemical weapons of war . Detailed studies of bacterial genes involved in the environmental degradation and recycling of a wide range naturally occurring and synthetic molecules show that degradation genes and degradation gene clusters play a major role in the worldwide carbon cycle.

Photosynthesis is considered the most important biological process on earth. And one of the most intensively studied photosynthetic organisms is the bacterium Rhodobacter sphaeroides. To start the research a local strain of R.sphaeroides, designated RS601, was isolated by Bill Tucker (my first australian PhD student) from a water sample obtained from a roadside ditch in Brisbane (Pemberton and Tucker,1977;Tucker and Pemberton,1978;1979;1980). One of the first discoveries made with this strain was lysogenic conversion to antibiotic resistance by a naturally occurring virus .(JM Pemberton, WT Tucker - Nature, 1977).

Subsequently when this strain was infected withe the broad host plasmid RP1 carrying the mecuric ion transposon Tn501 chromosome transfer occurred. This allowed the construction of the first genetic map of a photosynthetic bacterium(Pemberton and Bowen, J.Bacteriol, 1981). Mapping revealed that the photosynthesis gene cluster was on the main chromosome. Remarkably chromosome transfer occurred from a site right next to the photosynthesis gene cluster with early transfer of the entire cluster into the recipent cell. This provides a potential mechanism for the evolution and spread of photosynthesis genes. A clone bank of RS601 was constructed using pHC79:: Tn5deltaBamH1. This vector allowed cosmid cloning into the BamH1 site of Tn5. These Tn5 cosmid clones were transposed onto the broad host range plasmid pR751. The ability to transfer the entire cosmid clone bank to a wide range of bacteria led to the first cloning and heterologous expression of a carotenoid gene cluster (Pemberton&Harding,Current Microbiology,1986 & 1987).This indicated that genes involved in photosynthesis could be transferred to and expressed in a range of unrelated non-photosynthetic bacteria. Subsequent heterologous expression of carotenoid genes in an increasing variety of plants led to the production of foods enriched in the precursors of vitamin A e.g. Golden Rice (Erik Stokstad, Science Nov 20, 2019) . Vitamin A deficiency is the major preventable cause of blindness in children under 5 years of age; it affects up to 500,000 children each year. Using the same clone bank in mapping experiments in Rhodobacter sphaeroides I observed a few pale colonies in which carotenoid biosynthesis was suppressed. Subsequent detailed analysis of one of these cosmids led to the discovery of the long sought master regulator (PpsR) of bacterial photosynthesis and provided the first detailed insight into the mechanism by which bacterial photosynthesis is regulated at the molecular level (A Gene from the Photosynthetic Gene Cluster of Rhodobacter sphaeroides Induces trans Suppression of Bacteriochlorophyll and Carotenoid Levels in R.sphaeroides and R.capsulatus (R.J.Penfold and JM Pemberton, Current Microbiology, 1991; Sequencing, Chromosomal Inactivation and Functional Expression in E.coli of ppsR a Gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides. RJ Penfold and JM Pemberton. J.Bacteriol May 1994).Early studies by Cohen-Bazire, Sistrom and Stanier (1957) revealed that oxygen and blue light had varying effects on photosynthesis in Rhodobacter. The effect of oxygen was profound. The effect of blue light was more muted. The initial sequencing of ppsR (Penfold and Pemberton, 1994) revealed the presence of only two cys residues suggesting a possible mechanism for the profound effect of oxygen on PpsR repressor activity. Studies of conformational changes/repressor activity of PpsR in the presence and absence of oxygen have produced mixed results(Gomelsky et al.,2000;Masuda and Bauer.,2002). In contrast the muted effect of blue light on photosynthesis appears to be due to the blue light sensitive, anti-repressor AppA. (Gomelsky and Kaplan,1995). It is not known if any other environmental signals modulate PpsR activity.The rhodobacter research led to the construction of pJP5603 which allowed the precise insertion of a defined segment of DNA into a bacterial genome (Penfold and Pemberton,1992 ; Zordan,Beliveau,Trow,Craig and Cormack, 2015). The technique was used to either add functional genes or groups of genes to a precise location in the genome or to precisely target and inactivate individual genes. The site of insertion/mutagenesis is tagged with an antibiotic resistance gene. This process is known as “recombineering” ( Zhang et al., 1988). As with all forms of mutagenesis there are “off target” mutations. The consequences of such ”off target” mutations can range from minimal to extensive.

In a study of a range of genes encoding secreted enzymes involved in the degradation of naturally-occurring biological polymers e.g xylanases, cellulases,amylases, chitinases etc I attempted to obtain secretion genes from Chromobacterium violaceum. Again using the pHC79:: Tn5deltaBamH1 vector used in the study of the photosynthesis genes (Pemberton&Harding,Current Microbiology,1986 & 1987) I constructed a cosmid clone bank of C.violaceum. The clone bank I constructed did not produce secretion genes but instead 2-3 of the clones expressed the intense purple pigmented violacein in E.coli(Pemberton,1986). Subsequent subcloning revealed the gene cluster occupied 8kb and transposon mutagenesis revealed intense blue and intense green intermediates. (Pemberton et.al.,Current Microbiology,1991). I am grateful to Trudy Grossman for the detailed study of this cluster which included sequence analysis and functional characterisation of the violacein biosynthetic pathway (August et al., 2000). The functional analysis of the violacein gene cluster revealed that VioA VioC and VioD belong to the PheA(phenol) /TfdB (2,4-D) group of FAD dependant mono-oxygenases. TfdB is encoded by the 2,4-D degradation gene cluster of the broad host range IncP plasmid pJP4 carried by Ralstonia eutropha JMP134. This provides a link between the degradation of a man-made molecule-2,4-D and the synthesis of an anti-tumour antibiotic-violacein. Remarkably, under certain circumstances this 2,4-D degradation pathway can convert 2,4-D into the well known plant antibiotic-protoanemonin (Blasco,R et al., 1995).In 1983 Burt Ensley , Barry Ratzkin and co-workers (Ensley et al.,Science,1983) discovered that the naphthalene dioxygenase gene from Pseudomonas putida enabled E.coli K12 to synthesise the famous blue dye indigo from tryptophan; a second gene, VioD, from the violacein gene cluster also enabled E.coli K12 to produce indigo (Cheah et al.,Acta Crystallographica,1998). Further studies using the violacein gene cluster led to the development of techniques and vectors that should allow cloning and stable, high level expression of more antibiotic biosynthesis pathways in E.coli K12, particularly pathways from the prolific antibiotic producers the Streptomycetes providing novel antibiotics in the fight against antibiotic resistant pathogens (Sarovich and Pemberton,2007; Philip,Sarovich and Pemberton,2008 & 2009;Ahmetagic & Pemberton, 2010 & 2011;Ahmetagic, Philip ,Sarovich,Kluver and Pemberton,2011).An article published in June 2013 by Stevens and co-workers PLoS ONE 8(5) showed that a native gene cluster from Streptomyces rimosus encoding tetracycline can be directly expressed in E.coli K12.

For the first time researchers have showed the expression of the violacein gene cluster in a eukaryote-the yeast Saccharomyces cerevisiae (Lee et al., 2013). Such a discovery may indicate that the violacein gene cluster can be expressed in organisms which range from microbes to man. It may also indicate that major pathways from microorganisms can be engineered and expressed in a range of eukaryotes. Since violacein is a potent anticancer agent it is of interest to determine if the violacein cluster engineered into bacteria of the microbiome of an animal reduces cancer rates. Alternatively it may be possible to engineer the violacein pathway directly into an animal and observe if cancer rates are reduced. In view of the purported prokaryotic ancestry of eukaryoyic organelles such as mitochondria and chloroplasts ,one possible way of boosting violacein synthesis in eukaryotic cells could be to integrate the violacein gene cluster into organelle DNA.

Finally, violacein is chemically related to the well known anti-cancer drug staurosporine and possesses anticancer, antifungal, anti-parasite, antibacterial and antiviral activities;it might be possible to synthesise structural variants of violacein with more potent activity against various cancers and drug/antibiotic resistant pathogens. Interestingly is now known that violacein producing bacteria associated with the skin microbiome of certain frogs provides some protection against extinction by the worldwide spread of ‘chytrid’ fungus(Harris et.al., 2009). In addition, frogs have been used in cancer studies and may provide a simple model to test the anticancer properties of violacein. Since the violacein gene cluster is expressed in a wide range of bacteria ( Dr D S Philip, personal communication;D.S/Philip.PhD Thesis 2010) and has potent activity against the malarial parasite Plasmodium falciparum and other mosquito borne parasites, there is the possibility that mosquitoes engineered to carry the violacein gene cluster might be resistant to parasite infection. The cluster could be stably incorporated in the genomes of bacteria normally inhabiting the surface or the gut of the mosquito.A recent patent application (United States Patent Application 20170280730) indicates that Chromobacterium introduced into the microbiome of mosquitoes is useful for the prevention of transmission of malaria and dengue virus.In addition, chemical modification of violacein may produce drugs with even higher levels of activity against parasites including the malarial parasite ( Wilkinson et al., 2020). Violacein has activity against the pandemic virus Covid19 and there is some knowledge of its mode of action (Duran et al., 2021). Testing may reveal if it has activity against both Kappa and Delta Covid19 variants.Violacein can inhibit infection by HIV and COVID (Doganci et al., 2022)

• Fellow, American Society for Microbiology
• Fellow, Australian Society for Microbiology

Selected Publications:

• Ahmetagic, Adnan, Philip, Daniel S., Sarovich, Derek S., Kluver, Daniel W. and Pemberton, John M. (2011) Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12. Plasmid, 66 3: 152-158. doi:10.1016/j.plasmid.2011.07.006
• Ahmetagic, Adnan and Pemberton, John M. (2011) Antibiotic resistant mutants of Escherichia coli K12 show increases in heterologous gene expression. Plasmid, 65 1: 51-57. doi:10.1016/j.plasmid.2010.11.004
• Ahmetagic, Adnan and Pemberton, John M. (2010) Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12. Plasmid, 63 2: 79-85. doi:10.1016/j.plasmid.2009.11.004
• Philip, Daniel S., Sarovich, Derek S. and Pemberton, John M. (2009) Complete sequence and analysis of the stability functions of pPSX, a vector that allows stable cloning and expression of Streptomycete genes in Escherichia coli K12. Plasmid, 62 1: 39-43. doi:10.1016/j.plasmid.2009.03.002