Etude du système ars bactérien de résistance à l'arsenic : diversité des transporteurs d'arsénite et analyse moléculaire d'un opéron ars

Abstract : Arsenic is an ubiquitous toxic metalloid. The two most common forms of arsenic in the environment are inorganic arsenate [As(V)] and arsenite [As(III)]. Many microorganisms are able to transform arsenic (by oxidation, reduction and methylation) and thus play an important role in the biogeochemical cycle of this metalloid. Among the mechanisms of arsenic transformation, the ars system seems to be widely distributed in bacteria. This detoxification system involves the reduction of As(V) in As(III) (the most toxic and most mobile form) which is then pumped out of the cell through an integral membrane transporter. Two unrelated families of arsenite transporters have been described in prokaryotes: the well-characterized ArsB family and the Acr3p family which is subdivided into two subgroups. In this study, we designed three sets of degenerate primers allowing the amplification of the arsB and ACR3 genes. These primers were used to screen a collection of forty-one arsenic-resistant strains isolated from two soil samples from Lorraine with contrasting levels of arsenic concentration. PCR results showed that 70.7 % of the isolates contained a gene related to arsB or ACR3 family. The ACR3 genotype was predominant over arsB. These results validated the use of our primers for the identification of arsenite transporter genes. In addition to validating the primers, we characterised a novel collection of forty-one arsenic-resistant strains: molecular identification, MIC determination for As(III), As(V) and Sb(III) and detection of arsenite oxidase and arsenate reductase activities. Among these forty-one strains, arsenite oxidase activity was detected in two strains and arsenate reductase activity in thirty-nine. The Microbacterium sp. A33 isolate showed high resistance level to both As(III) and As(V). We used a molecular approach to identify arsenic resistance (ars) genes of this strain. Our analysis revealed an original system composed of 6 genes : arsT, arsX, ACR3(1), arsRC2, arsC1 et arsC3 that encode a thioredoxin reductase, a thioredoxin, an arsenite transporter, a fusion protein "regulator-arsenate reductase", an arsenate reductase and another arsenate reductase respectively. The first five genes belong to the same operon and arsC3 gene is transcribed in the opposite direction. Thioredoxin and thioredoxin reductase are essential to thioredoxin-coupled arsenate reductase activity. However, the majority of ars operons don?t encode these proteins. The atypical protein ArsRC2 is also rarely present in ars operon. The ArsC3 arsenate reductase is another particularity of Microbacterium sp. ars system. ArsC3 contains the CX5R specific motif of thioredoxin-coupled arsenate reductases but don?t contains in the C-terminal extremity the two cysteine residues essential for reduction of As(III) to As(V). A strategy is proposed to analyze the role of each Microbacterium sp. ars gene in resistance to As (III) and As(V).
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Asma Achour-Rokbani. Etude du système ars bactérien de résistance à l'arsenic : diversité des transporteurs d'arsénite et analyse moléculaire d'un opéron ars. Médecine humaine et pathologie. Université Paul Verlaine - Metz, 2008. Français. ⟨NNT : 2008METZ010S⟩. ⟨tel-01752573⟩

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