Brunella Perito

Bacillus subtilis Gene Cluster Involved in Calcium Carbonate Biomineralization

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-beta-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.

Molecular basis of bacterial calcium carbonate precipitation.

Calcium carbonate precipitation is a widespread process, occurring in different bacterial taxonomic groups and in different environments, at a scale ranging from the microscopic one of cells to that of geological formations. It has relevant implications in natural processes and has great potentiality in numerous applications. For these reasons, bacterial precipitation has been investigated extensively both in natural environments and under laboratory conditions. Different mechanisms of bacterial involvement in precipitation have been proposed. There is an agreement that the phenomenon can be influenced by the environmental physicochemical conditions and it is correlated both to the metabolic activity and the cell surface structures of microorganisms. Nevertheless, the role played by bacteria in calcium mineralization remains a matter of debate. This chapter reviews the main mechanisms of the process with particular focus on what is known on molecular aspects, and discusses the significance of the precipitation event also from an evolutionary point of view.

Effect of different extraction methods on fiber quality of nettle (Urtica dioica L.)

Chemical extraction, water retting, microbiological and enzymatic methods were applied on entire nettle stalks and/or unretted decorticated fiber of a selected fiber nettle clone. Morphological and mechanical properties and chemical composition were then determined on fiber samples. The first interesting result concerned the good degree of separation between fibers and shives obtained by mechanical scutching applied on stalks stored for 1 year, probably resulting from natural retting processes occurring during the storage. Microbiological retting (anaerobic plus aerobic bacteria) of entire stalks and/or unretted decorticated fiber produced fibers with a higher quality than water retting. Both enzymes used (Viscozyme ® L and Pectinex® Ultra SP-L), improved fiber quality if EDTA was added. The enzyme vat retting gave good results on both water-retted fibers and unretted decorticated fibers, while the spray enzyme treated fibers usually displayed thicker diameter, lower cellulose content and, for Viscozyme ® L, lower strength values, without differences between the two storage methods used after enzyme application.

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.

Characterization and sequence analysis of a Streptomyces rochei A2 endoglucanase-encoding gene

A 7-kb fragment of Streptomyces rochei A2 chromosomal DNA was cloned into pAT153 and shown to confer endoglucanase (EglS) activity on Escherichia coli cells. In E. coli clones, the EglS was secreted into the periplasm. Deletion analysis revealed that an 827-bp fragment was enough for the enzymatic activity. Sequence analysis showed that the 827-bp fragment codes for the catalytic domain of the enzyme. The complete sequence of the gene (eglS) is 1149-bp long. A signal peptide, a catalytic domain and a cellulose-binding domain were identified from the nucleotide sequence, and the EglS found to belong to the family H of cellulase catalytic domains. These conclusions were substantiated by determination of the N-terminal sequence of the purified protein and zymogram analysis, which revealed protein species with a molecular mass equal to that deduced from the nt sequence analysis.

Antibacterial activity of larval saliva of the European paper wasp Polistes dominulus (Hymenoptera, Vespidae)

Summary.Microbiological tests demonstrated antibacterial activity (against Bacillus subtilis, Gram +, and Escherichia coli, Gram −) of larval salivary secretions of Polistes dominulus but failed to demonstrate its antifungal activity.

Exploitation of bacterial pectinolytic strains for improvement of hemp water retting

SummaryRetting is the major limitation to an efficient production of textile hemp fibres. Traditional retting has been carried out by autochthonous bacterial community. Aerobic and anaerobic pectinolytic strains were isolated from hemp or flax sources and characterised. Anaerobic pectinolytic strains had a wide range of acid polygalacturonase (PG) activity, whereas aerobic isolates did not produce any acid PG activity, but only an alkalophylic one, suggesting they could play a minor role in the retting process, except in the early stages. Analysis of 16S rDNA sequences assigned anaerobic strains to the Clostridium genus and aerobic isolates to the Bacillus and Paenibacillus genus. C. felsineum and C. acetobutylicum were confirmed as the main anaerobic agents. Nevertheless, a high proportion of anaerobic and aerobic pectinolytic strains was assigned to C. saccharobutylicum and B. pumilus, respectively, both species never being described as involved in water retting. Anaerobic and aerobic strains with high PG activity were selected and characterized. PG activity is well correlated with the strain retting efficiency and improvement of the process was obtained by inoculating the retting water with spores of selected aerobic and anaerobic bacteria. An advisable feature of retting strains is the absence of cellulosolytic activity. An aerobic strain with no cellulosolytic activity was identified. In contrast, all the anaerobic isolates showed cellulosolytic activity. Mutagenesis was ineffective for selection of Cel-Pec+ mutants. Localization of the C. felsineum L1/6 PG activity was investigated.

Bacterial Genes Involved in Calcite Crystal Precipitation

The natural precipitation of calcium carbonate crystals by bacteria has been proposed for conservative interventions in monument restoration, even if the use of heterotrophic viable organisms does not always seem appropriate for this purpose. In fact, chemical reactions with stone minerals due to metabolic by-products and the growth of fungi, due to the application of organic nutrients for bacterial development, can have negative effects on stone surfaces. We have studied crystal precipitation in a laboratory strain of Bacillus subtilis. The effectiveness of this process has been analyzed on small bioclastic limestone samples (Pietra di Lecce), determining their total porosity, amount of water absorbed and superficial cohesion. To overcome some of the possible problems involved with the use of viable bacterial cells, we have investigated the genetic mechanisms that control calcite precipitation. For this reason, we have selected Bacillus subtilis mutants that do not precipitate or which form calcite crystals faster. The data obtained suggest the presence of several genes involved in crystal formation.

Antibacterial activity of silver nanoparticles obtained by pulsed laser ablation in pure water and in chloride solution

Summary Silver nanoparticles (AgNPs) have increasingly gained importance as antibacterial agents with applications in several fields due to their strong, broad-range antimicrobial properties. AgNP synthesis by pulsed laser ablation in liquid (PLAL) permits the preparation of stable Ag colloids in pure solvents without capping or stabilizing agents, producing AgNPs more suitable for biomedical applications than those prepared with common, wet chemical preparation techniques. To date, only a few investigations into the antimicrobial effect of AgNPs produced by PLAL have been performed. These have mainly been performed by ablation in water with nanosecond pulse widths. We previously observed a strong surface-enhanced Raman scattering (SERS) signal from such AgNPs by “activating” the NP surface by the addition of a small quantity of LiCl to the colloid. Such surface effects could also influence the antimicrobial activity of the NPs. Their activity, on the other hand, could also be affected by other parameters linked to the ablation conditions, such as the pulse width. The antibacterial activity of AgNPs was evaluated for NPs obtained either by nanosecond (ns) or picosecond (ps) PLAL using a 1064 nm ablation wavelength, in pure water or in LiCl aqueous solution, with Escherichia coli and Bacillus subtilis as references for Gram-negative and Gram-positive bacteria, respectively. In all cases, AgNPs with an average diameter less than 10 nm were obtained, which has been shown in previous works to be the most effective size for bactericidal activity. The measured zeta-potential values were very negative, indicating excellent long-term colloidal stability. Antibacterial activity was observed against both microorganisms for the four AgNP formulations, but the ps-ablated nanoparticles were shown to more effectively inhibit the growth of both microorganisms. Moreover, LiCl modified AgNPs were the most effective, showing minimum inhibitory concentration (MIC) values in a restricted range of 1.0–3.7 µg/mL. An explanation is proposed for this result based on the increased surface reactivity of the metal surface due to the presence of positively charged active sites.

Cloning and expression in Escherichia coli of a Streptomyces β-glucosidase gene

Abstract Streptomyces A2 is a cellulolytic actinomycete isolated from the gut of termites. This microorganism secretes enzymes capable of degrading cellulose, which can be used as carbon source. A gene bank from Streptomyces A2 has been constructed in the Escherichia coli plasmid pUC 18 and screened for cellulase activities on agar plates. Two plasmids showing β-glucosidase activity were isolated and characterized. The restriction map of the plasmids showed that both carried the same DNA fragment containing the β-glucosidase gene. Transcription appeared to start from the plasmid lac promoter. The enzymatic activity was measured in cell extracts from E. coli cells carrying the plasmids. The length of the gene and the size of the coded protein were also determined.