The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology.

Recent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling. Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic-mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozyme-achromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others.

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12.

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to 'graze' on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan "plaque" formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.

Antibiotic resistant mutants of Escherichia coli K12 show increases in heterologous gene expression.

Using a variety of antibiotics, it was found that nine separate isolates of spontaneous antibiotic resistant mutants of Escherichia coli K12 pPSX-vioABCDE overproduce the anti-tumour antibiotic violacein. Subsequent analysis showed that seven of these mutations occurred on the plasmid pPSX-vioABCDE. The other two overproducing strains carried spontaneous chromosomal mutations to lincomycin and kanamycin. The kanamycin resistant mutant of E. coli K12 DH10B (AA23) and a lincomycin resistant mutant of E. coli K12 LE392 (AA24) increased the synthesis of violacein. The plasmid pPSX-vioABCDE opv-1 contains a violacein over-production (opv-1) mutation which when introduced into either E. coli K12 AA23 or AA24, resulted in a hyper-production of violacein. Remarkably, E. coli K12 AA23 pPSX-vioABCDE opv-1 produced 41 times the normal level of violacein. In addition, both E. coli K12 AA23 and E. coli K12 AA24 demonstrated an increase in expression of an alpha amylase gene from Streptomyces lividans and the urease gene cluster from Klebsiella oxytoca. These results suggest that selection of antibiotic resistant mutants can increase heterologous gene expression in E. coli K12. Additionally, the increased expression is a general effect applicable to genes and gene clusters cloned into E. coli K12 from both Gram-positive and Gram-negative bacteria.

Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12.

Previous studies showed that when pPSX-vioABCDE was used to transform E. coli K12 DH5alpha the strain retained the plasmid even after 100 generations of unselected growth but produced a low level of the anti-tumour antibiotic violacein. Markedly higher levels of violacein synthesis were obtained from E. coli K12 DH5alpha pUC18-vioABCDE and Sphingomonas sp. JMP4092 pPSX-vioABCDE. Unfortunately, both strains were extremely unstable regardless of presence or absence of antibiotic selection to retain the plasmid. The current study was undertaken to determine if strains of E. coli K12 could be isolated which stably over produce violacein. When a range of E. coli K12 strains were transformed with pPSX-vioABCDE, most produced small amounts of violacein. However, a small number of related strains of E. coli K12 JM101, JM105 and JM109 not only over-produced violacein, but also maintained the high stability. In addition, E. coli K12 JM109 strongly expressed an alpha amylase gene (amyA) from Streptomyces lividans indicating that the S. lividans amyA promoter is highly active in E. coli K12 JM109. In another set of experiments, a violacein overproduction mutation (opv-1) of the plasmid pPSX-vioABCDE was isolated which enabled E. coli K12 DH5alpha to overproduce violacein while retaining high stability. The plasmid pPSX-vioABCDEopv-1 possesses a single base pair deletion in the promoter region of the violacein operon. By combining the over producing strain E. coli K12 JM109 and the over producing plasmid pPSX-vioABCDEopv-1, a stable hyper producing strain (E. coli K12 JM109 pPSX-vioABCDEopv-1) was constructed. Finally, two additional stable vectors, pPSX10 and pPSX20, were constructed to facilitate subcloning and functional analysis studies.