Unit: Bacterial Genome Plasticity

Director: MAZEL Didier

We study the mechanisms responsible of the bacterial genome variability, with a special interest for those involved in exogenous gene acquisition - the horizontal gene transfer. Our model system is the integron, a natural genetic engineering system implicated in the development and dissemination of antibiotic resistance genes among Gram-negative species. We are also investigating other factors involved in genome plasticity of Vibrio species genomes, which are all constituted of two circular chromosomes that could have distinct dynamic characteristics.

A- the integrons.

Thirty years of molecular genetics, and in particular the advance of the "genomic era" over the last ten years, have shown that the acquisition of exogenous genetic material or lateral gene transfer, plays a primary role in bacterial evolution.

Perhaps, the most striking example of the impact of gene exchange has been the development of multi-drug resistance over the past 50 years. In this global phenomenon, the contribution of a particular class of mobile elements, the integrons, has been essential among Gram-negative bacteria. We are studying different aspects of this gene capture system: their distribution, their contribution to the adaptive capacity of their host and their recombination processes.

This natural genetic engineering system is composed of two basic elements: a gene coding an integrase of the site-specific tyrosine recombinase family and a primary recombination site, attI. The integrase activity allows the insertion of open reading frames, in the form of a circular cassette, at the recombination site. The tandem integration of cassettes leads to the assembly of multiple-resistance structures and integrons carrying up to 8 cassettes have been characterized. To date, more than 70 different resistance cassettes have been identified, allowing bacteria to evade most presently employed antibacterial agents. All these cassettes are composed of a single gene associated with a recombination site, the attC site (or 59-base element), indispensable for integrase recognition and recombination with attI. In general the cassette-associated genes have no resident promoter but recruit one from the integron following integration.

Five different classes of multi-resistance integrons (MRI) have been described as of today, based on their integrase gene sequence. They are all carried by mobile DNA elements (transposons and plasmids).

We have recently discovered another type of integron, the super-integron (SI), in the genome of a large number of γ-proteobacterial species. These integrons share several characteristics with MRIs, but they differ from them in 3 respects: i) their size (several tens to several hundred cassettes), ii) the strong similarity observed between the attC sites of the cassettes belonging to a specific SI and iii) the fact that most identified SI cassettes are not linked to antibiotic resistance.

The structural differences existing between the MRIs and the SIs led us to propose that they evolved from SIs through the entrapment of SI integrase genes and their cognate attI sites by mobile DNA elements. The subsequent harvesting of cassettes from various SI sources then led to the establishment of contemporary MRIs, through the application of a constraint: the antibiotics used by man.

This year we have focused our work on three objectives:

1) Establishing the structural characteristics of SIs in Vibrio. More particularly we have concentrated our efforts on establishing an inventory of those cassettes encoding poison-antidote systems.

We observed the presence of multiple cassettes carrying post-segregational killing (PSK or poison-antidote) systems related to those found on plasmids or phage, in all the SIs characterized so far. For example, the V. cholerae N16961 SI contains 5 different families of PSK in 9 cassettes spread along the entire cassette array. The function of these cassettes in the SIs is still unclear. These cassettes may play an important role in stabilizing these massive arrays of independently mobile genetic units by regulating the levels of random deletion through homologous recombination between repeated cassettes, since a probability exists to lose the PSK cassettes along with neighboring cassettes, an event which would result in cell death,.

We have already demonstrated the functionality of several of the PSK cassettes (the ccdAB cassette from the V. fischeri SI, the phd-doc from V. metschnikovii and V. cholerae) in E. coli but also in their original strain. We also demonstrated that these cassettes carried a strong promoter allowing high levels of expression (in collaboration with Laurence Van Melderen, ULB, Belgium).

2) New genetic tools devoted to the Vibrio genome manipulation.

In order to carry out our different projects we have constructed and developed several new tools which allow easy disruption of targeted genes in the different Vibrio species that we study. This has lead to two publications.

3) Nature of the recombination partners and of the IntI recombination substrates.

The structural differences between MRIs and SIs led us to compare the dynamic parameters of recombination, and the specificity of the different types of integron integrases. By comparison to the other site-specific recombination systems that have been studied, understanding of the integron recombination processes is still superficial and many questions remain to be answered. For example, is the attC site homogeneity observed in the SI cassette array due to a narrower specificity of the SI integrase compared to the MRI integrase for which a low specificity toward the attC site sequence recombination has been demonstrated ?

We attempted to answer this question by a comparative study of the IntI1 (class 1 MRI integrase) and VchIntIA (V. cholerae SI integrase). The results of this study are now in press in the Journal of Bacteriology.

B-. Sequencing the V. lentus MEL 32 genome.

The second project is to investigate other factors involved in genome plasticity of the complex genome of Vibrio species. The Vibrio group includes a large number of pathogenic species whose hosts range from human to aquatic animals. The few species so far characterized have been found to carry two circular chromosomes. Despite an apparent long evolutionary history of this partition in two molecules, the comparison of chromosome content in the related cholerae and parahaemolyticus species indicates a high variability. The selective advantage conferred by such an organization is unknown. This also raises a number of fundamental questions, in particular for chromosome segregation, and offers a unique opportunity to experimentally manipulate chromosomes.

To increase our knowledge, we initiated in collaboration with C. Bouchier (sequencing platform 1, Institut Pasteur) the genome sequencing of a Vibrio species, which is only remotely related to the Vibrio species sequenced so far. Our strain, V. lentus MEL 32 is an oyster pathogen, which has been responsible for high mortality rates in oyster beds in France since 1991.

V. lentus is only remotely related to V. cholerae and V. vulnificus, and belongs to the other major branch in the Vibrio radiation, which also contains V. fischeri and V. salmonicida (a fish pathogen). It is very likely that the data collected from this genome project will allow a better understanding of the evolutionary constraints exerted on the Vibrio genomes as a whole and will give new clues to understand the partition of the two chromosomes in this bacterial group, as well as understanding the specific pathogenic properties of this oyster pathogen.

Keywords: integron, site-specific recombination, horizontal gene transfer, evolution, Vibrio, cholerae

Activity Reports 2004 - Institut Pasteur

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