Friday, June 26, 2009

A rapid method to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection

First Gene Cassettes of Integrons as Targets in Finding Adaptive Genes in Metagenomes
Lionel Huang, Christine Cagnon, Pierre Caumette, and Robert Duran
Applied and Environmental Microbiology, 2009, 75(11):3823–3825

Here, I will introduce a short publication focusing on a very narrow specific problem. This paper propose a rapid method for the selection of clones carrying an integron first gene cassette that is useful for finding adaptive genes in environmental metagenomic libraries.

Integrons are genetic structures capable of capturing and excising gene cassettes, which usually encode adaptive proteins in different environmental contexts, such as genes for degradation of pollutants, and resistance to antibiotics or heavy metals. Thus, environmental pressures may favor the propagation of cassettes conferring a selective advantage.

Integrons are not a new and hot topic, but there is an increasing interest in finding of adaptive genes associated with integrons among the huge metagenomes. The integration of a new gene cassette, catalyzed by the integrase, occurs by recombination between the attC site and the attI site of the integron. The first gene cassette of an integron is, therefore, the last one integrated. In previous studies, the determinations of gene cassette collection from environmental metagenomes did not target first gene cassettes, since they were performed by PCR methods targeting attC sites. As the first gene cassette is the closest gene to the promoter, its expression level is the highest in the integron. Thus, it is a good target to find new adaptive genes in metagenomes.

The method was developed by using a pure strain isolate, Xanthomonas campestris ATCC 33913T, which carry an integron possessing 23 gene cassettes. One environmental sample, coastal sediment, was used to validate the method. There are two key points in developing this method, one is the construction of first gene cassettes libraries. To amplify the first gene cassettes, a forward primer targeting the intI gene or attI site must be used. Forward primer AJH72 was used for PCR of X. campestris DNA, and primer ICC48 (intB-inverted primer), targeting the class 1 integron intI, was used for PCR of sediment metagenome. A less-degenerated primer ICC21, was designed to target the attC sites from class 1 and 2 integrons. The other key point is the trick for clone selection. Due to the particular structure of the attC site with inverted repeat sequences, the reverse primer was also used in the forward direction. As a result, several amplified fragments were obtained, and the sequence analyses revealed that most of them were gene cassettes other than the first one. Here, the authors develop a triplex PCR method by labeling the forward primer with HEX (6-carboxyhexafluorescein). The fluorescent PCR fragments were selected for further sequencing.

This method was then applied to coastal mud metagenomes, and 23 fluorescent fragments were detected and sequenced. A total of 29 open reading frames (ORF) were characterized as potentially transcribed by an integron promoter. The first-gene cassettes of integrons appear to be good candidates to find gene cassettes, which aid bacteria in effecting a rapid adaptive response. We are now able to reveal integron last gene acquisitions of environmental bacterial communities submitted to stressful conditions. The PCR method combined with the screening method leads to 100% of clones carrying a first gene cassette. Thus, this new method allows the focus to be on spreading first gene cassettes in metagenomes after a specific stress.

In this paper, the authors propose a good idea to construct gene cassette libraries enriched with first gene cassettes and an associated screening method for the clone selection. However, the only one drawback in this paper is that the relationship between the function of ORF and the oil degradation should be discussed further. Since the first gene cassettes was enriched by adding oil into the coastal sediment, it should be served as the environmental pressure for selecting adaptive genes. I have checked the information from EMBL, where the author deposited the sequences, but still no related information in the database.

Hui Li, PhD
University of Idaho

Friday, June 19, 2009

DNA transfer proteins of broad-host-range plasmid can mediate chromosomal DNA transfer

The R1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome. Richard Meyer (2009) J. Bacteriol. 191: 1574-1580

It is well-known that plasmids mediate horizontal gene transfer among bacteria and play major roles in the rapid spread of antibiotic resistance. Generally, plasmid-mediated horizontal gene transfer from one bacterial chromosome to another requires the help of transposons as follows; in the first step, a transposon that has captured chromosomal genes moves into the plasmid; in the second step, the plasmid moves into a recipient cell by conjugative transfer; and in last step the transposon on the plasmid jumps into the chromosome of the recipient cell.

This paper showed that the above-described scheme is not the only way that plasmids can participate in the horizontal transfer of chromosomal genes. The author found that a plasmid can directly transfer donor chromosomal DNA into the recipient chromosome.

In a canonical model, conjugative DNA transfer starts with the DNA cleavage at the origin of transfer "oriT", a unique site on the plasmid, which is mediated by a protein called "relaxase". Previously, the author found that relaxase of the broad-host-range plasmid R1162 (also called RSF1010) can initiate DNA transfer at several sequence variants of oriTR1161. The observed promiscuous activity of the relaxase raised the possibility that plasmids can directly mediate the transfer of chromosomal DNA from oriT-like sequences in a chromosome (Jandal S. and Meyer R., 2006).

Draper et al. (2005) showed that relaxase can mediate recombination between two directly repeated oriTs on the transferred DNA in the recipient cell. This nature of the relaxase was used in the experiment to test the hypothesis that relaxase can initiate DNA transfer from cryptic oriT in a chromosome. A plasmid that has the original oriT and a selectable drug-resistance gene marker was artificially integrated into the downstream region of one of the candidate oriT sites in the chromosome of donor strain Pectobacterium atrosepticum to make directly repeated oriT sites. If single-strand DNA is branched out from the candidate oriT and moves into recipient cells, the transferred DNA would become a plasmid that carries a hybrid oriT comprised of the candidate oriT and the original oriT due to the recombination activity of the relaxase. As the author expected, the plasmid that has a part of the donor chromosome and a hybrid oriT was obtained in the recipient E. coli. This result indicates that the initiation of transfer does happen at the candidate oriT in P. atrosepticum.

The next questions are "What length of DNA is it possible to transfer?" and "Is the transferred chromosomal DNA integrated into chromosome in the recipient cell?" To answer these questions, the author used an E. coli strain as donor strains, that has a drug-resistance gene marker at a particular location in the chromosome. In the presence of helper plasmids that express relaxase and pillus proteins, the drug resistance gene marker was transferred and integrated into the chromosome of recipient E. coli. The candidate oriT closest to the resistance gene was 40 kbp away from the resistance gene in the donor chromosome. It suggests that a chromosomal DNA fragment of at least 40 kbp was transferred in the mating process. Surprisingly, even when the closest oriT was eliminated from the donor chromosome, the transfer of the drug resistance maker was observed with almost the same frequency as it was in the presence of the closest candidate oriT. This result suggests that chromosome transfer can be initiated at multiple cryptic oriT sites in the donor chromosome. Given that the second closest candidate oriT is 708 kbp away from the resistance gene, a DNA fragment of at least 708 kbp was indicated to be transferable in this experiment.

The authors estimated that there are 10 candidate oriT sites in the P. atrosepticum chromosome and 8 in the E. coli chromosome, which could be active in the presence of R1162 relaxase. Although it is still not clear how many plasmids have a potential to mobilize fragments of the chromosome, this article clearly showed that there is a novel manner of horizontal gene transfer.

Bacteriophage are also known to mediate the transfer of host's DNA in the manner called "general transduction", where host's DNA are accidentally packed in phage's capsid and are introduced into new host cells. Relaxase-mediated chromosomal DNA transfer resembles phages' general transduction, but different in that the length of transferable DNA is not limited in the relaxase-mediated chromosomal DNA transfer; the size of transferable DNA is limited in general transduction due to the limited size of phages' capsid. Given the size of transferable DNA, it seems that plasmids play much more important roles in bacterial evolution than bacteriophages.


Meyer R. (2009) The R1162 mob proteins can promote conjugative transfer from cryptic origins in the bacterial chromosome. J. Bacteriol. 191: 1574-1580

Jandle S. and Meyer R. (2006) Stringent and relaxed recognition of oriT by related systems for plasmid mobilization; implications for horizontal gene transfer. J. Bacteriol. 188: 499-506

Draper O., César C. E., Machón C., de la Cruz F., and Llosa M. (2005) Site-specific recombinase and integrase activities of a conjugative relaxase in recipient cells. Proc. Natl. Acad. Sci. USA 102: 16385–16390.

H.Yano. University of Idaho