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