Friday, August 21, 2009

Need we optimize the mating system when deal with every different genus?

Assessment of horizontal gene transfer in Lactic acid bacteria – A comparison of mating techniques with a view to optimising conjugation conditions
Niamh Toomey, Áine Monaghan, Séamus Fanning, Declan J. Bolton
Journal of Microbiological Methods, 2009, 77: 23–28

The most common laboratory techniques used to assess HGT in vitro include: plate, filter and broth mating protocols. While there is a general acceptance that higher transfer frequencies occur when using solid-phase mating mediums (since conjugation requires mating cells to be in close contact with each other), few studies have experimentally compared HGT techniques, with a view to standardizing these approaches, thereby providing meaningful direct comparisons.
In this study, plate, filter and broth mating techniques were assessed and compared by using a taxonomically diverse group, Lactic acid bacteria (LAB). LAB is a Gram-positive, catalase-negative group, which share the capacity to ferment sugars into lactic acid. Due to their aerotolerant anaerobic nature they are found in a variety of different environments. LAB also has the potential to act as resistance gene reservoirs with the ability to transfer these genes in a range of different environments; including transfer to pathogenic species. Genes conferring resistance to antimicrobials such as tetracycline, erythromycin, streptomycin, chloramphenicol, and vancomycin, have been found in LAB isolated from foods. These resistance genes were found to be located on transferable elements including plasmids and conjugative transposons. In present study, one L. lactis strain with the broad-host range plasmid pAMβ-1 [containing an erythromycin resistance marker, erm(B)], and two L. lactis strains with the conjugative transposon Tn916 [expressing a tetracycline resistance gene, tet(M)] were used as the donor strains, along with a marked (Strr, Rifr) L. lactis strain as recipient.
The plate mating technique used in this study is almost the same with that used in TopLab, except that following overnight incubation and scraping of the bacterial spots, additional medium was used to wash the plate for accurately calculating the transfer frequency. The filter mating technique is exactly the same with that we used. The broth mating method is just to add equal volume donor culture and recipient culture into one tube, and then following the overnight incubation, directly dilute the mixed culture and spread onto selective plates. Transconjugants were confirmed as the true transconjugants but not the reverted mutants by using antibiotic selection, E-tests to determine MICs, PCR assays to detect the corresponding marker genes, DNA fingerprinting by pulsed-field gel electrophoresis (PFGE), and Southern blotting.
Based on the results of transfer frequency, the general trend is plate > filter > broth. In addition, in most cases, there is no significant difference between plate mating and filter mating. Effects of different pH, varying from 6.0 to 8.0, on the transfer rate were also detected. The pH of medium between 6.0 and 7.0 had no significant effect on transfer rate, while at pH 8.0 the conjugation was completely inhibited.
As the author said, this paper is the first study to examine the influence of mating protocols on both plasmid and transposon conjugal transfer between lactococcal strains. The results provide the detailed information about which system should be recommended in future LAB HGT studies.
However, some of the other research works are not in agreement with the results from this study. Both Lampkowska et al. (2008) and Langella et al. (1996) suggested that the filter facilitates a greater degree of donor-recipient contact than the plate method. Therefore, a question rises up. Need we optimize the mating system (including the mating techniques and environmental factors, such as temperature, pH) when we deal with every different genus, such as Pseudomonas, Agrobacterium, and Cupriavidus, which currently used in our lab? If it does, then a great amount of work we should do. Do you think it is necessary? Feel free to take part in the discussion. It is a Blog, and there should be some discussion here.

Additional reference:

Lampkowska, J., Feld, L., Monaghan, A., Toomey, N., Schjørring, S., Jacobsen, B., van der Voet, H., Andersen, S.R., Bolton, D., Aarts, H., Krogfelt, K.A., Wilcks, A., Bardowski, J., 2008. A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species. Int. J. Food Microbiol. 127, 172–175.

Langella, P., Zagorec, M., Ehrlich, S.D., Morel-deville, F., 1996. Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake. FEMS Microbiol. Lett. 139, 51–56.

Hui Li, PhD
University of Idaho

Sunday, August 16, 2009

The defective prophage pool of Escherichia coli O157: Prophage-prophage interactions potentiate horizontal transfer of virulence determinants

Asadulghani M. and Ogura Y. et al, (2009) PLOS Pathogens 5: 1-15.

Bacteriophage is one of the major genetic elements promoting horizontal gene transfer between bacteria. Enterohemorrhagic Escherichia coli O157:H7 (Sakai isolate) contains eighteen prophages in its genome (Ohnishi et al. 1999). Two of the eighteen prophages carry Shiga toxin gene clusters; stx1AB and stx2AB; these gene products kill eukaryotic cells by inhibiting protein synthesis. Interestingly, all prophages in O157 have mutations in genes that encode basic phage functions such as site-specific recombination, replication, cell lysis, or structural proteins constituting the head and tail. Evidence obtained by genome analysis suggests that each prophage is defective. Thus, one might think this strain is not very problematic. But, it may be not true.

To assess the role of defective prophages in horizontal gene transfer of virulence determinants, the authors analyzed each basic function of the prophages in O157: excision from chromosome, phage DNA amplification in response to DNA damage, ability to be released as phage particles, and infection of new recipient cells.

Among eighteen prophages, eight prophages including the stx gene-containing phages were found to be released as phage particles, and among them four were able to infect both or either of the two E. coli recipient strains tested. These results strongly suggest that prophages in O157 are complementing defective functions with each other to make practically transferable phages.

Importantly, the phage particle produced often contains a chimeric DNA fragment made of two different prophages' fragments, indicating the recombination of prophages in host cells. Although the newly generated phage DNA were not extensively sequenced in this study, it is possible that some phage fragments reconstituted intact, functional phage through recombination.

This paper nicely explains the way bacteriophages live and propagate in a host. I think this paper is a good example of a post-genomic sequence study. In this era, we can understand how mobile genetic elements are acting in a population by combining sequence analysis and standard genetic experiments. I suspect that plasmids and transposons are also repeating cycles of inactivation and activation as shown for these phages. They will never be extinguished as long as similar mobile genetic elements exist in the world.

Additional reference:
Ohnishi M. and Tanaka C. et al., (1991) DNA Res. 6: 361-368

Posted by H. Yano, Univerisity of Idaho