Factors affecting formation of large calcite crystals (≥1mm) in Bacillus subtilis 168 biofilm

B4 is the most common medium used in general organomineralization studies and has been used to assay or to characterize mineral precipitation potential. In an exercise for the optimization of the laboratory conditions of crystal precipitation in vitro, we used Bacillus subtilis 168 as a type strain and its isogenic mutants. While literature is mainly focused on observing generic precipitation, we investigated the requirement to obtain large crystals (≥1mm), which could be advantageous in wide-ranging implications for bioconsolidation of soil, sand, stone, and cementitious materials. Calcite crystals are visible on B4 agar plates within 7 days at 37˚C after inoculum of B. subtilis 168 strain. In this study we show that to form large crystals with a diameter ≥1mm several conditions must be met: i) Reduced amount of B4 medium into the Petri plate improve crystal formation. 55mm Petri plates contained only 4mL of B4 agar medium reached a plateau in 6 days at 37ºC. High moisture and presence of water condense would decrease crystal formation. ii) Inoculation of cells using a rod instead of a circular shaped spot. When the same number of B. subtilis cells was streaked, rod-shape biofilm significantly fostered crystal precipitation, while spot-shape prevented precipitation. iii) When more than one biofilm is present within the same plate, mutual interactions can affect precipitation in each biofilm. iv) Spherical nucleation sites are identified as initial step during the formation of large calcite crystal

A method to assess bioavailability of antibiotics in anthropogenic polluted ecosystems by using a bacterial fitness test

Antibiotics released in the environment exert a selective pressure on the resident microbiota. It is well accepted that the mere measurement of antibiotics does not reflect the actual bioavailability. In fact, antibiotics can be adsorbed or complexed to particles and/or chemicals in water and soil. Bioavailable concentrations of antibiotics in soil and water are subjected to great uncertainty, therefore biological assays are increasingly recognized as that allow an indirect determination of the residual antibiotic activity. Here we propose how a fitness test for bacteria can be used to qualitatively assess the bioavailability of a specific antibiotic in the environment. The findings show that by using a pair of resistant and sensitive bacterial strains, the resulting fitness can indirectly reflect antibiotic bioavailability. Hence, this test can be used as a complementary assay to other biological and chemical tests to assess bioavailability of antibiotics

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

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

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

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

Effect of anthropogenic pollution on the fitness of tetracycline sensitive Shigella flexneri in Thames river water

Urban rivers may be source of antibiotics contamination that could support spread of antibiotic resistant bacteria (ARB) to the population. It is important to understand to what extent the presence of pollutants in urban rivers influences fitness of ARB. In an exercise to estimate this contribution, microcosms were generated from Thames river (London, UK) from different locations: upstream and downstream the city center. The concentration of the polycyclic aromatic hydrocarbons (PAHs) benzo(a)pyrene, pyrene and phenantrene was found to be 128, 171 and 128 times higher in downstream sector when compared to upstream sector, respectively. Filtered microcosms for each sector were enriched with tetracycline at lethal (10 μg/mL) and sub-lethal (10 ng/mL) concentrations and the fitness of an isogenic pair of Shigella flexneri 2a YSH6000 (tetR) and S. flexneri 2a 1363 (tetS) was then measured. In the presence of selective pressure in upstream microcosms, the resistant strain outcompeted the sensitive one, as expected. In contrast, sensitive S. flexneri tetS was found to significantly compete with resistant S. flexneri tetR at lethal concentrations of tetracycline in downstream microcosms, where levels of PAHs were the highest. Further experiments showed that PAHs rendered the resistant S. flexneri tetR ∼20% more sensitive to tetracycline. Sensitive S. flexneri tetS strain was able to persist at lethal concentration of tetracycline in downstream microcosms, at higher concentrations of PAHs. Our findings suggest that in a polluted river sensitive S. flexneri cells may still thrive in presence of selective pressure. Fitness tests provide an additional tool to measure bioavailability

In vivo induction of neutrophils chemotaxis by secretory aspartyl proteinases of Candida albicans

Secretory aspartyl proteinases (Saps) of Candida albicans are key virulence traits which cause inflammasome-dependent, aseptic inflammation in a mouse model of vaginitis. In this paper, neutrophil migration in response to Sap2, Sap6 and chemo-attractive products released from Sap-treated vaginal epithelium was measured in vitro, ex vivo and in vivo. Our results show that Sap2 and Sap6 induce neutrophil migration and production of potent chemoattractive chemokines such as IL-8 and MIP-2 by vaginal epithelial cells. Our data suggest that at least part of MIP-2 production depends upon IL-1β activity. The vaginal fluid of Candida-infected mice contained a heat-labile inhibitor of neutrophil candidacidal activity that was absent from the vaginal fluid of Sap-treated mice. Overall, our data provide additional information on the capacity of C. albicans Saps to cause aseptic vaginal inflammation and highlight the potential role of some chemokines released from vaginal epithelial cells in this phenomenon