Wednesday, July 14, 2010

Fosmid cloning: Alive and kicking

Although advances in next-generation sequencing technology have replaced the need for clone libraries in many laboratories, fosmid libraries are still useful in a variety of functional genomics studies.

Xu et al.1 present the first report of a host-specific restriction system associated with S-modification of DNA (phosphorothioation), instead of methylation. The authors observed that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolated from E. coli, while protecting its own DNA by site-specific phosphorothioation. They located the gene in S. enterica by screening a genomic library created using the CopyControl™ Fosmid Library Production kit. The authors screened the fosmid libraries using degenerate gene cluster–specific primers to locate the genes responsible for phosphorothioation and also to perform mutational analysis. Their results establish a biological role for phosphorothioation activity, and suggest that DNA S-modification may act not only as protective system against bacteriophage infection, but also as an epigenetic signal for new biological functions.

In another study by Lucker et al.2, the CopyControl Kit was used to reconstruct the complete genome of Candidatus Nitrospira defluvii (Ca. N. defluvii) from a metagenomic fosmid library prepared from an activated sludge enrichment culture. The immense ecological and technical significance of Nitrospira contrasts with the scarce knowledge about these bacteria. Except for one 137-kb contig, genomic sequences from Nitrospira have not been obtained yet. This situation has been highly unsatisfactory because deeper insight into the biology of these elusive nitrate oxidizing bacteria is crucial for a better understanding of nitrogen cycling in natural and engineered systems. On the basis of this first-deciphered Nitrospira genome and experimental data, the authors show that Ca. N. defluvii differs fundamentally in its enzymatic repertoire and metabolic pathways from all other known nitrifying bacteria. The current study provides valuable insights into the evolution of nitrite oxidation.

ResearchBlogging.org1. Xu, T. et al. (2010). A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella Nucleic Acids Research DOI: 10.1093/nar/gkq610
2. Lucker, S. et al. (2010). A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1003860107

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