Physiological requirements for carbonate precipitation during biofilm development of Bacillus subtilis etfA mutant

Although the implications of calcium carbonate (CaCO(3)) precipitation by microorganisms in natural environments are quite relevant, the physiology and genetics of this phenomenon are poorly understood. We have chosen Bacillus subtilis 168 as our model to study which physiological aspects are associated with CaCO(3) (calcite) formation during biofilm development when grown on precipitation medium. A B. subtilis eftA mutant named FBC5 impaired in calcite precipitation was used for comparative studies. Our results demonstrate that inactivation of etfA causes a decrease in the pH of the precipitation medium during biofilm development. Further analysis demonstrated that eftA extrudes an excess of 0.7 mol H(+) L(-1) with respect to B. subtilis 168 strain. Using media buffered at different pH values, we were able to control calcite formation. Because etfA encodes the alpha-subunit of a putative flavoprotein involved in fatty acid metabolism, we compared the intracellular levels of NADH among strains. Our physiological assay showed that FBC5 accumulated up to 32 times more NADH than the wild-type strain. We propose that the accumulation of NADH causes a deregulation in the generation of the proton motive force (DeltamicroH(+)) in FBC5 producing the acidification

Bacillus subtilis fadB (ysiB) gene encodes an enoyl-CoA hydratase

Fatty acids are essential components of membranes and are an important source of metabolic energy. In bacteria, the β-oxidation pathway is well known in Escherichia coli. Bacillus subtilis possesses a considerable number of genes, organized in five operons, that are most likely involved in the β-oxidation of fatty acids. Among these genes, only one product, FadRBs (YsiA), has been recently characterized as a transcriptional regulatory protein which negatively regulates the expression of β-oxidation genes including those belong- ing to the lcfA operon, including fadRBs (ysiA). The probable involvement of the FadRBs (YsiA) regulon members in β- oxidation is inferred from data based on BLASTP similarity of their gene products. In this work, we report the cloning and the expression of B. subtilis fadBBs(ysiB), belonging to the lcfA operon, and the functional characterization of its product as an enoyl-CoA hydratase, demonstrating the actual involvement of these genes in fatty acid β-oxidation

A Bacillus subtilis cell fraction (BCF) inducing calcium carbonate precipitation: biotechnological perspectives for monumental stone reinforcement

Abstract Monumental stone decay is a consequence of the weathering action of physical, chemical and biological factors, which induce a progressive increase in porosity. To cope this degradation, bacterial calcium carbonate mineralization has been proposed as a tool for the conservation of monumental calcareous stones. The advantage of this kind of treatment is to obtain a mineral product similar to the stone substrate, mimicking the natural process responsible for stone formation. In this work, the possibility to induce CaCO3 mineralization by a bacteria-mediated system in absence of viable cells was investigated and tested on stone. Our results showed that Bacillus subtilis dead cells as wells as its bacterial cell wall fraction (BCF) can act as calcite crystallization nuclei in solution. BCF consolidating capability was further tested in laboratory on slab stones, and in situ on the Angera Church, a valuable 6th century monumental site. New crystals formation was observed inside pores and significant decrease in water absorption (up to 16.7%) in BCF treated samples. A little cohesion increase was observed in the treated area of the Angera Church, showing the potential of this application, even though further improvements are needed

Bacterial community analysis on the Mediaeval stained glass window "Natività" in the Florence Cathedral

Microbial corrosion of glass causes problems on delicates antique glass samples. Until now, the effect of microbial activity on corrosion phenomena has not been well documented. Only a few studies have been published concerning the microflora growing on glass surfaces. The present study deals with the characterization of cultivable aerobic bacteria isolated from the historical glass window "Natività" in the Florence Cathedral, designed by Paolo Uccello and realized by Angelo Lippi between 1443 and 1444. Microbial strains were sampled from four of the 25 panels of the "Natività" in the occasion of a recent conservation treatment, due to the presence of various kinds of crusts. One hundred microorganisms were isolated, about 50% bacteria and 50% fungi. Bacteria were submitted to morphological characterization and classified in the Gram group. For twenty strains, from different glass panels, the 16S rDNA gene was amplified and sequenced. Sequence analysis showed genus Bacillus, Arthrobacter and Paenibacillus as the most representative. In particular Bacillus and Paenibacillus are crusts associated. Phylogenetic relationship among isolates was determined. Chemical analysis of the glass and crusts completed the study

Black microcolonial fungi as deteriogens of two famous marble statues in Florence, Italy

Blackened areas on outdoor marble statues are a significant esthetic problem due to the presence of deteriorating agents. Microcolonial black fungi, which have their natural ecological niche on rocks, play an important role in deterioration of stones used in monuments, such as marble and limestone. Black fungi were isolated from two very valuable statues exposed to the outdoor environment in Florence, Italy, the "Ratto delle Sabineâ" and the "Copia del David", and these fungi were demonstrated to be responsible for the blackening areas on the statues. The black strains showed many features common to members of rock-inhabitants dematiaceous fungi. Morphological and molecular characterization, including phylogenetic analysis, indicated that the strains isolated from both statues and in different times belong to the same species and can be assigned to the rock-inhabitant genera Sarcinomyces and Phaeococcomyces. Red yeasts growing in close proximity to the black ones, with no visible effect on the statues, were also characterized on the morphological and molecular level and identified as Sporobolomyces yunnanensis

Impacts of Anthropogenic Pollutants on Benthic Prokaryotic Communities in Mediterranean Touristic Ports

Ports and marinas are central nodes in transport network and play a strategic role in coastal development. They receive pollution from land-based sources, marine traffic and port infrastructures on one side and constitute a potential pollution source for the adjacent coastal areas on the other. The aim of the present study was to evaluate the effects of organic and inorganic co-contamination on the prokaryotic communities in sediments from three Mediterranean ports. The structure and composition of the bacterial and archaeal communities were assessed by targeted metagenomic analysis of the 16S rRNA gene, and the links of prokaryotic communities with environmental and pollution variables were investigated. The harbors presented pronounced site-specificity in the environmental properties and pollution status. Consistently, the structure of archaeal and bacterial communities in surface sediments exhibited a strong spatial variation among the three investigated ports. On the contrary, a wide overlap in composition of prokaryotic assemblages among sites was found, but local variation in the community composition and loss of prokaryotic diversity was highlighted in a heavily impacted port sector near a shipyard. We provided evidences that organic matter, metals and PAHs as well as temperature and salinity play a strong role in structuring benthic bacterial communities significantly contributing to the understanding of their responses to anthropogenic perturbations in marine coastal areas. Among metals, copper was recognized as strongly associated with the observed changes in bacterial assemblages. Overall, this study provides the first assessment of the effects exerted by multiple organic and inorganic contaminations on benthic prokaryotes in ports over a large spatial scale and designates bacterial community as a candidate tool for the monitoring of the sediment quality status in harbors

Black microcolonial fungi as deteriogens of two famous marble statues in Florence, Italy

Blackened areas on outdoor marble statues are a significant esthetic problem due to the presence of deteriorating agents. Microcolonial black fungi, which have their natural ecological niche on rocks, play an important role in deterioration of stones used in monuments, such as marble and limestone. Black fungi were isolated from two very valuable statues exposed to the outdoor environment in Florence, Italy, the "Ratto delle Sabineâ" and the "Copia del David", and these fungi were demonstrated to be responsible for the blackening areas on the statues. The black strains showed many features common to members of rock-inhabitants dematiaceous fungi. Morphological and molecular characterization, including phylogenetic analysis, indicated that the strains isolated from both statues and in different times belong to the same species and can be assigned to the rock-inhabitant genera Sarcinomyces and Phaeococcomyces. Red yeasts growing in close proximity to the black ones, with no visible effect on the statues, were also characterized on the morphological and molecular level and identified as Sporobolomyces yunnanensis