Technology

Recently the full genome of a Streptomyces (S. Seasidensis) was fully sequenced by Stenz (Stenz et al 2009). The (linear) chromosome is 6,667,507 b. p. long with a GC-content of 73.1 and is predicted to contain approximately 6,825 protein encoding genes.

The Streptomyces are gram positive bacteria, with high GC content. They are found in soil and are responsible for the production of most of the commercially available antimicrobial, antifungal and immunosuppressant substances. Thus, they are very suitable hosts for the expression and secretion of eukaryotic gene products.

The discovery in the meantime of the novel antibiotic Brightonomycin named after Brighton et al,2008 has brought new hope for the treatment of MRSA infections, which are causing a wide concern in hospital settings worldwide. Brighton and coworkers have described Brightonomycin, as a natural occurring polyketide, produced by strains of Streptomyces seasidensis in vitro. This natural antimicrobial belongs in the Macrolide family and is similar to Erythromycin in that is it is a 14-membered lactone ring with ten asymmetric centers and two sugars, bearing a L- mycarose in place of erythromycins  L-cladinose. (Mycarose is an 2,6-didesoxy-3-C-methyl-L-ribohexose, while cladinose is a 3-methyl ether.)

Brightonomycin is produced in the stationary phase of Streptomyces cycle of life. Attempts to identify metabolic pathways for the action and synthesis of Brightonomycin are now being carried out nation and worldwide but results have not yet been published. Brighton and colleagues however, have managed to demonstrate its bactericidal activity against strains of Methicillin resistant Staphylococcus Aureus, esp. strains EMRSA15 and EMRSA16, that are resistant to both Erythromycin and Ciprofloxacin.

AIM The aim of this project will be to identify the genes responsible for Brightonomycin production, a novel antibiotic that is potent against Methicillin Resistant Staphylococcus Aureus, in Streptomyces seasidensis. The purpose of the responsible gene identification will be to promote the knowledge on the location and function of the gene(s), the characterization and study of the referring protein families and metabolic pathways required for the production of Brightonomycin .The final goal of this project will be the production, isolation and testing of a suitable strain of S.seasidensis with increased Brightonomycin production for use in the pharmaceutical industry, as a novel antimicrobial against MRSA infections.

MATERIALS AND METHODS

Strains of S.seasidensis that will be used in this study have been isolated from the Laboratory for Genomic Research, (previously published work).

Instruments that are going to be used in this project will come from the variety of our state of the art genomic laboratory. Resources of the laboratory include
BioAnalyzer (Agilent) Lab-on-a-chip platform designed to provide improved accuracy and reproducibility analysis of DNA, RNA, proteins and cells.

NanoDrop Spectrophotometer for highly accurate analyses of 1 micro liter samples of nucleic acids.
Sequence Detection System (SDS) TaqMan 7700 and TaqMan 7500 (Applied Biosystems) 96-wells plate real-time PCR instruments.

StepOne (Applied Biosystems) 48-wells real-time PCR instrument.
Microlab Duo (Hamilton) Pipetting robot.
Clondiag ATR 01 Array Tube Reader.
Agilent G2539 High resolution micro array scanner.
ABI 3130xl Automated 16 capillaries DNA sequencer.
Illumina-Solexa Genome Analyzer II high-throughput sequencer.
Hewlett-Packard  Agilent 1100 UV HPLC system.

MOLECULAR GENOMIC AND PROTEOMIC INVESTIGATIONS

Until now, 3 different strains of S.seasidensis with different Brightonomycin production capabilities have been isolated in our department
Strain A-119 (parent strain) is the strain sequenced from the genomic mapping project and has been initially isolated from a mountain soil region in Chuxiong, Yunnan, China.
Strain B-22 (100-fold production of brightonomycin) and
Strain C-43L (mutant strain- no production of brightonomycin), both have been the result of high through-put gene trapping efforts by previous research in our department.

Production of the strains In more detail, we have previously used an electroporation experiment of a Tn5-derived transposon-transposase enzyme complex into S.seasidensis, which resulted in the creation of random insertion mutant strains.These were screened phenotypically to identify the strain(s) that exhibited altered brightonomycin production (from none to excessive). Strain C-43L exhibited no production of brightonomycin that was demonstrated by lack of the drug detection on silica gel chromatography and HPLC. Strain B22 produced 100-fold more brightonomycin. The full method for brightonomycin isolation and detection is discussed in the next section in detail.

Isolation of a multi-copy plasmid (PXR-B22), responsible for the over stimulation of brightonomycin production Using the phage lambda Red recombination system, we have created multiple multi-copy plasmids from strain-B22, which has the ability to produce 100-fold more brightonomycin compared to the parent strain. Further electroporation on parent strains (A-119), has led to the correct identification of a multi-copy plasmid (PXR-B22) containing a DNA fragment from the S.seasidensis genome that stimulates the over production of brightonomycin, as demonstrated by HPLC detection.

PROCEDURES FOR GENE IDENTIFICATION

The instruments employed in this project, i.e. the brightonomycin production gene identification, are all described in the methods section. All experiments will be carried in enriched precursor media for antibiotic production (acetate, glucose). In detail, experiments will include the following procedures

1) Identification and description of the genomic DNA inside the multi copy plasmid, by extraction and amplification-sequencing using PCR (Taqman SDS). By excluding all essential for replication material that exist in large quantities in multi copy versus single copy plasmids, we aim to describe base by base the gDNA inside the isolated plasmid PXR-B22. Comparison in large databases of genes (i.e.BLAST) and plasmid libraries will help us identify the purpose of the plasmid gene. The full genome map of S.seasidensis is going to prove useful for the location of the above gene in the genetic material of the Streptomyces.

2) Detailed search for the presence of the multi-copy plasmid gDNA in our 3 S.seasidensis strains. This will be facilitated by PCR-sequencing, by first creating a primer from the original plasmids gDNA. Detection of its presence is an essential step in determining and establishing the exact gene location in the B-22 strain. Also, in the parent strain (a-119) it is anticipated to have the same location and function, while in strain C-43L we expect not to locate any of the non-essential for plasmid replication gene material.

3) Electroporation of the plasmid inside all three available strains and recording of the occurring mutations and phenotypes

B-22 strainParent strainC-43L strainPlasmidPhenotype (Brightonomycin production)100-fold1-foldNone-( suppression, induction, further induction, nothing)

4). This identification will be done using a comparative micro array scanning procedure on the 3 strains- targeted at the regions missing in the mutant C-43L. This will hopefully lead to the identification of candidate genes for the production of the antibiotic brightonomycin. In order to prove such a relationship, we will have to perform multiple confirmation analyses (mutations by homologous recombination of alleles) followed by phenotypical measurements and HPLC assays. In comparison and simultaneous analysis of the strains micro array results and the genome of the Streptomyces and predicted protein products (see genome project protein families), one has the possibility to identify the cluster of genes for production of the drug itself, the promoter gene location or different regulatory proteins genes (for example global regulatory proteins and small regulatory molecules e.g. AfsA  a diffusible signaling molecule) which are all possible targets in the observed over stimulation on production

5) Comparison of the best candidate genes with the plasmid genomic DNA to determine which is its function (i.e. gene promoteractivator activator for regulatory protein).This will aid in the establishment of whether this particular plasmid is sufficient for antibiotic production in large scales. It is possible that micro arrays and the step by step comparison to the genome, will identify novel promoter areas, capable of being copied in even swifter to use low-copy plasmids, for the activation of antibiotic synthesis in 1000-fold or even more quantities.

Theoretical knowledge exists from the fascinating research currently being conducted in Streptomyces sp. to allow for high copy number plasmids to incur the over stimulation of Polyketide production (Fong et al 2007). Hence we believe that although time consuming, this project has the potential to achieve its goal which is the characterization of the genetic mechanisms behind the production of brightonomycin and proposed means for its overproduction. Further studies (protein specific analysis) will be required to study the biological and metabolic pathways that compliment this effort.