Giorgio Mastromei

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.

Effects of selected pectinolytic bacterial strains on water-retting of hemp and fibre properties.

Aims:  To study the effect of selected bacterial strains on hemp water‐retting and properties of retted fibre.

Preliminary results of tests facing with the controlled retting of hemp

Abstract An important component of the implementation of the hemp crop management is the microbiological retting of the straw. At the Experimental Institute for Industrial Crops, research has been focused on this topic and in 1997 trials of retting performed in tanks placed in the laboratory or in open field were carried out. Cultivar ‘Fibranova’ was employed, harvested at the time of full flowering. For the laboratory tests, 12 plastic tanks containing well water were used. One of these was taken as the control and each of the remaining 11 tanks were inoculated with different strains of Clostridium felsineum. Four different retting times were experienced (3, 6, 9 and 12 days). The fibre was assessed for the morphological, chemical and physico-mechanical properties. The retting in open field was performed in four concrete tanks; (1) in pond water in an open tank, (2) in pond water in a tank covered with a transparent plastic film (to raise the temperature of the water), (3) in well water in an open tank and with the addition of a culture of C. felsineum, and (4) in well water in an open tank, without the addition of C. felsineum. Six increasing retting durations were adopted (2, 4, 6, 8, 10 and 12 days). The fibre obtained was evaluated taking into account colour, softness and fineness. The results obtained indicated that the water type, the temperature of retting water and the bacterial inoculum all influenced the duration of the process and the quality of the fibre. Retting was completed in 6 days in pond water and in 12 days in well water. Raising the temperature of water from 24 to 28°C reduced the duration of the process from 6 to 4 days. It was found that the bacterial strains ‘L1–6’ and ‘C1–6’ of C. felsineum reduced the duration of the retting process from 12 to 6 days (with a water temperature of 20°C), leading to a clearer, softer and finer fibre.

Construction of a new vector conferring methotrexate resistance in Nicotiana tabacum plants

A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.

Molecular epidemiological investigation of an outbreak of Pseudomonas aeruginosa infection in an SCT unit

From May to October 2006, six severe Pseudomonas aeruginosa infections were diagnosed in patients undergoing SCT in the SCT unit of the Careggi hospital (Florence, Italy). Four of the infected patients were treated consecutively in the same room (room N). On the hypothesis of a possible environmental source of infection, samples were collected from different sites that had potential for cross-contamination throughout the SCT unit, including the electrolytic chloroxidant disinfectant used for hand washing (Irgasan) and the disinfectant used for facilities cleaning. Four of the environmental samples were positive for P. aeruginosa: three Irgansan soap samples and a tap swab sample from the staff cleaning and dressing room. The AFLP (amplified fragment length polymorphism) typing method employed to evaluate strain clonality showed that the isolates from the patients who had shared the same room and an isolate from Irgasan soap had a significant molecular similarity (dice index higher than 0.93). After adequate control measures, no subsequent environmental sample proved positive for P. aeruginosa. These data strongly support the hypothesis of the clonal origin of the infective strains and suggest an environmental source of infection. The AFLP method was fast enough to allow a ‘real-time’ monitoring of the outbreak, permitting additional preventive measures.

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.

Molecular Characterization of Acinetobacter Isolates Collected in Intensive Care Units of Six Hospitals in Florence, Italy, during a 3-Year Surveillance Program: a Population Structure Analysis

ABSTRACT The strain diversity and the population structure of nosocomial Acinetobacter isolated from patients admitted to different hospitals in Florence, Italy, during a 3-year surveillance program, were investigated by amplified fragment length polymorphism (AFLP). The majority of isolates (84.5%) were identified as A. baumannii, confirming this species as the most common hospital Acinetobacter. Three very distinct A. baumannii clonal groups (A1, A2, and A3) were defined. The A1 isolates appeared to be genetically related to the well-characterized European EU II clone. A2 was responsible for three outbreaks which occurred in two intensive care units. Space/time population dynamic analysis showed that A1 and A2 were successful nosocomial clones. Most of the A. baumannnii isolates were imipenem resistant. The genetic determinants of carbapenem resistance were investigated by multiplex PCR, showing that resistance, independently of hospital origin, period of isolation, or clonal group, was associated with the presence of a bla OXA-58-like gene and with ISAba2 and ISAba3 elements flanking this gene. bla OXA-58 appeared to be horizontally transferred. This study showed that the high discriminatory power of AFLP is useful for identification and typing of nosocomial Acinetobacter isolates. Moreover the use of AFLP in a real-time surveillance program allowed us the recognition of clinically relevant and widespread clones and their monitoring in hospital settings. The correlation between clone diffusion, imipenem resistance, and the presence of the bla OXA-58-like gene is discussed.

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.