Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological implications. Nevertheless, little is known of the molecular mechanisms by which bacteria foster calcium carbonate mineralization. In our laboratory, we are studying calcite formation by Bacillus subtilis, in order to identify genes involved in the biomineralization process. A previous screening of UV mutants and of more than one thousand mutants obtained from the European B. subtilis Functional Analysis project allowed us to isolate strains altered in the precipitation phenotype. Starting from these results, we focused our attention on a cluster of five genes (lcfA, ysiA, ysiB, etfB, and etfA) called the lcfA operon. By insertional mutagenesis, mutant strains carrying each of the five genes were produced. All of them, with the exception of the strain carrying the mutated lcfA operon, were unable to form calcite crystals. By placing transcription under IPTG (isopropyl-␤-D-thiogalactopyranoside) control, the last gene, etfA, was identified as essential for the precipitation process. To verify cotranscription in the lcfA operon, reverse transcription-PCR experiments were performed and overlapping retrocotranscripts were found comprising three adjacent genes. The genes have putative functions linked to fatty acid metabolism. A link between calcium precipitation and fatty acid metabolism is suggested.

Abandoned tailing dumps from mining industry represent important sources of metal contamination in the surrounding environments. This study evaluates the potential of two Mediterranean native plants, Pistacia lentiscus and Phragmites australis, for phytoremediation of two Sardinian contaminated mine sites. A 6 months study has been conducted at greenhouse-controlled conditions with the aim of investigating the plant capability to tolerate high metal concentrations and to extract or immobilize them within the roots. The possibility to mitigate stress on the plants and improve treatment efficiency by adding compost as amendment was also evaluated. Both species were able to restrict accumulation of Cd, Pb and Zn to the root tissues exhibiting a metal concentration ratio of plant roots to soil bioavailable fraction higher than two (four in the case of Zn). However, the two species showed different adaptation responses, being the survival of P. australis after 6 months in contaminated soil lower (25 %-58 %) than that observed for P. lentiscus (77 %-100 %). Compost addition resulted in a lower metal uptake in tissues of both plants and a higher survival of P. australis, whilst almost no effect was observed as regard the growth of both species. The two tested species appear to be promising candidates for phytostabilization, P. lentiscus exhibiting a greater adaptability to heavy metal contaminated matrices than P. australis.

SummaryPectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilis T and B. pumilus T .

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