Tuesday, March 31, 2009

A novel gene module protecting bacteria from phage infection

The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS, and Salmond GP. (2009) Proc Natl Acad Sci U S A. 106:894-899.

Recently it has become easy to determine the complete sequence of 100 kb long plasmids. However, the ensuing annotation process, assigning a function to a DNA sequence, is still a frustrating process for people who are working on sequence analysis; often, the newly sequenced DNA segment does not show homology to well-known genes which makes difficult to judge whether or not there is a gene in the segment.

Let's hope that no homology is a good sign for a big discovery. Here, I introduce a discovery of the novel gene module on Erwinia carotovora plasmid pECA1039, which protect host bacterium from phage infection.

Erwinia carotovora is a plant pathogenic bacterium that causes rot in diverse vegetables. The author's group has been studying virulence mechanisms of E. carotovora (Barnard A.M. and Bowden S.D. et al., 2007). They identified a probable protein coding sequence, named toxN, through the complete sequence analysis of E. carotovora's 5,620-bp cryptic plasmid., whose product shows 31% amino-acid identity to a protein associated with phage abortive infection (Abi) in gram-positive bacteria (Emond E. and Shirley E.D. et al., 1998). Genes related to Abi generally exert a cellular process that shuts down the phage lytic cycle or that kills phage-infected cells to prevent the phage particle production (Chopin M.C. and Chopin A. et al., 2005). The toxN gene on pECA1039 is adjacent to a potential coding sequence toxI that includes five copies of a 36-bp sequence tandem repeat, followed by an inverted repeat sequence. The potential gene product ToxI exhibits no similarity to proteins in databases. Since the Abi system was not so common among gram-negative bacteria, the authors focused the study on the toxI-toxN region and experimentally showed that the toxI-toxN region confers phage-resistance to the host.

The authors have two major subject to be addressed: one is the mechanism of phage resistance, and another is whether or not toxI encodes a protein.

The authors showed that toxN encodes a toxic protein that inhibits the host's growth, which means that the mechanism of phage resistance might be a growth inhibition induced by phage infection. The authors also found that the co-expression of toxI with toxN can counteract the toxic activity of ToxN. But, the hypothetical protein ToxI seemed to not be produced from the toxI gene region according to Western blotting analysis, using the modified toxI gene fused with a sequence coding for hexa-histidine tag.

It is possible that toxI RNA itself has an activity to counteract ToxN. To test this hypothesis, the authors introduced a translational stop codon into the toxI coding sequence and found that the ToxN-counteracting activity was still retained in the mutant toxI region. Furthermore, point mutations that do not change the amino-acid sequence of ToxI but do change the transcript sequence resulted in the loss of ToxN-counteracting activity. These results suggest that the toxI RNA is responsible for the antitoxin function. They also pointed out that similar gene modules that are comprised of the tandem repeat region and the toxN homologous gene are present in diverse Eubacteria and Archea.

Detailed mechanisms of toxin (ToxN) induction and the interaction between the toxI RNA and the ToxN protein are still unclear. However, it is obvious that the authors identified a novel type of functional RNA. The authors' work proved that we can still discover new biological concepts from plasmid sequences.

Fineran PC, Blower TR, Foulds IJ, Humphreys DP, Lilley KS, Salmond GP. (2009) The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proc Natl Acad Sci U S A. 106:894-899.

Barnard AM, Bowden SD, Burr T, Coulthurst SJ, Monson RE, Salmond GP. (2007) Quorum sensing, virulence and secondary metabolite production in plant soft-rotting bacteria. Philos Trans R Soc Lond B Biol Sci. 362:1165-1183.

Chopin MC, Chopin A, Bidnenko E. (2005) Phage abortive infection in lactococci: variations on a theme. Curr Opin Microbiol 8:473-479.

Emond E, Dion E, Walker SA, Vedamuthu ER, Kondo JK, Moineau S. (1998) AbiQ, an abortive infection mechanism from Lactococcus lactis. Appl Environ Microbiol. 64:4748-4756.

posted by H. Yano (University of Idaho)

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