Sergio Casella

A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti.

Abstract. During free-living reproductive growth, Sinorhizobium meliloti accumulates poly-β-hydroxybutyrate (PHB) and glycogen, and produces and excretes exopolysaccharides and β-1,2-glucan. In previous investigations, PHB-minus mutants of S. meliloti 41 were obtained and studied; and the genes for PHB biosynthesis, phaAB and phaC, were described. In this work, the role of an open reading frame (orf) upstream of phaAB is studied. This orf is designated aniA because the gene was found to be expressed during anaerobic growth. Under low oxygen conditions, glycogen decreases and the production of extracellular polymeric substances (EPS) is partially repressed. When the aniA mutant is incubated under oxygen-limiting conditions, the only significant change observed is an overproduction of EPS. Subsequent in planta tests showed that although the mutant strain produced abundant nodules, only very low acetylene-reduction activity was detected, indicating that nitrogen fixation was not adequately supported by endogenous substrates.

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum.

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.

Aspects of Plant‐Microbe Interactions in Heavy Metal Polluted Soil

The role of soil microbiota, specifically rhizospheric microorganisms, in the development of phytoremediation techniques has to be elucidated in order to speed up the process and to optimize the rate of mobilization/absorption of pollutants. To meet such a purpose, several heavy metal resistant bacterial strains were isolated from a contaminated soil and from the rhizosphere of some spontaneous plants grown therein, such as Brassica sp., Trifolium repens, Trifolium pratense and Chenopodium album. Colonization of the rhizosphere is known to be helpful for bacteria, but their presence is also supposed to be functional to the plants, especially if in connection with their ability to produce Plant Growth Promoting (PGP) compounds (e.g. indole-acetic acid) or to protect the plant from pathogens. Therefore, in such an adverse environment, plants and bacteria surely take advantage by cooperating. Several bacterial isolates were tested and found to be capable of producing PGP compounds. Among them, a highly heavy metal resistant strain (Br-10) was isolated from the roots of a spontaneous plant belonging to the Brassicaceae family, whose members are known to accumulate heavy metals, and identified by phenotypic tests and 16S rDNA analysis as Serratia plymuthica. To evaluate its putative biocontrol activity, a screening of its antagonistic properties against various soil phytopathogens was also undertaken. Among the pathogenic fungi tested, a strain of Phytophtora megasperma f. sp. glycinea was found to be sensitive. Preliminary investigations of Br-10 resistance mechanisms, performed through hybridization studies on genomic DNA, suggested for S. plymuthica strain Br-10 the existence of a resistance mechanism different from that codified by the well-known czc gene cluster of Alcaligenes eutrophus that encodes for cadmium, zinc and cobalt resistance through an efflux system. By the use of Inductively Coupled Plasma analysis Cd was found to be preferentially adsorbed or accumulated rather than being forced out of the cell or not imported at all. Growth kinetics studies revealed that increasing Cd levels do not seem to affect cell growth up to the stationary phase, while having a clear impact on cell viability during the stationary phase.

Polyhydroxyalkanoates production by engineered Cupriavidus necator from waste material containing lactose

Cupriavidus necator DSM 545 is a well-known polyhydroxyalkanoates (PHAs) producer, but unable to grow on lactose. The aim of this study was to construct a recombinant strain of C. necator that can use lactose-containing waste material such as cheese whey, to produce PHAs. One of the intracellular PHA depolymerases (phaZ1) of C. necator was chosen to insert the lacZ, lacI and lacO genes of Escherichia coli. This would have the effect to allow polymer production on lactose and, at the same time, to remove part of the PHA intracellular degradation system. Disruption of phaZ1 was achieved by gene replacement after isolating a fragment of this gene and interrupting it with a cartridge containing the lac genes and a synthetic promoter. Growth and polymer production studies of the genetically modified (GM) strain mRePT in lactose, whey permeate and hydrolyzed whey permeate as carbon sources, were performed. Lower PHA degradation and higher yields were obtained compared to the wild-type strain. Inactivation of the putative depolymerase gene phaZ3 on mRePT recombinant strain was also reported.

Dimethyl carbonate and switchable anionic surfactants: two effective tools for the extraction of polyhydroxyalkanoates from microbial biomass

The availability of green and cheap technologies to recover polyhydroxyalkanoates (PHAs) from microbial biomass is crucial for the development of a reliable and sustainable production chain. Here, two novel protocols are proposed to extract PHAs from Cupriavidus necator. The first method is based on PHA-extraction with dimethyl carbonate (DMC), a green solvent that is completely biodegradable and less harmful to humans and the environment than most solvents. The procedure can be applied directly to concentrated microbial slurries or to dry biomass, affording very high polymer recovery (>85%) and excellent purity (>95%). No degradation/decomposition of the polymer is observed in both cases. The second protocol uses fatty acid carboxylates as surfactants, which disrupt cell membranes, providing excellent polymer recovery (>99%) and high purity (>90%). Ammonium laurate can be successfully used and easily recycled (98%) by lowering the pH through CO2 addition. Therefore, both protocols reported here are effective and sustainable: the recovery and purity of the obtained PHAs are very high, the use of toxic chemicals is avoided, and the recycling of various solvents/surfactants used in the processes is optimal.

Resuscitation of Viable But Not Culturable Sinorhizobium meliloti 41 pRP4-luc: Effects of Oxygen and Host Plant

A plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and stably inherited in Sinorhizobium meliloti 41 as a reporter gene. The strain obtained, S. meliloti 41/pRP4-luc, and its parental strain served as a model system for viable but not culturable (VBNC) resuscitation experiments in both in vitro and soil samples. Incubation under oxygen (O2) concentrations varying from 1% to atmospheric levels did not result in resuscitation. A demonstration of recovery was attained through exposure to the appropriate concentrations of antibiotics, bacteriostatic chloramphenicol, and bactericidal ampicillin. The resuscitation ratio was 1 recovered VBNC cell in every 105 5-cyano-2,3-di-4-tolyl-tetrazolium chloride (CTC+) bacteria. Although isolated VBNC rhizobia were unable to nodulate Medicago sativa, which apparently did not enhance VBNC reversion, resuscitated bacteria maintained their symbiotic properties. Soil experiments showed that the lack of O2 leads to onset of VBNC status as in liquid microcosm, but the number of recoverable and culturable cells decreased more drastically in soil.

Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium

The drought‐tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H. coronarium. Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, β‐galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species‐specific bacteriophages were isolated and a strain‐typing grid established. In order to characterize further and fingerprint the different Rhizobium ‘hedysari’ isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed‐field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.

Effect of various treatments on contents of adenine nucleotides and rna of mediterranean soils

Abstract Changes in adenine nucleotides (ATP, ADP and AMP) were investigated in soils subjected to various treatments and compared with bacterial and fungal counts. An organic and a clay soil stored for 3 yr at room temperature showed low ATP concentrations (0.36 and 0.26 nmol g −1 , respectively) which markedly increased after moistening both soils; this indicates that the prolonged storage did not eliminate the capacity of ATP synthesis by these soils; in the organic soil bacteria and fungi were unaffected by moistening while only bacterial counts were increased in the clay soil. By moistening soil with NaN 3 solution the ATP increase was annulled in the organic soil and only reduced in the clay soil. Air-drying a sandy clay-loam soil resulted in a 40% drop in ATP while microbial counts were unaffected; rewetting the air-dried soil to 50% of the water holding capacity (WHC) did not increase ATP. Decrease in the ATP content was more marked when moistened or rewetted soils rather than fresh or airdried soils were fumigated; the fumigation of soils moistened with the NaN 3 solution generally decreased the ATP concentration. Sodium azide generally decreased recoveries of ATP, ADP and AMP especially in the organic soil; the effect of the inhibitor on enzymatic conversion of AMP to ADP and of ADP to ATP was also investigated in the soil extracts. Adenylate energy charge (AEC) values of moist field samples were 0.65, 0.70 and 0.80 for clay, sandy-clay loam and organic soils, respectively. Air-drying moist soils caused a marked drop in ATP and RNA and an increase in ADP, AMP and total adenine nucleotides. The AEC value of CHC1 3 fumigated fresh moist soil were very low ranging from 0.06 to 0.1 thus indicating a large prevalance of dead cells even if the bacterial numbers by plate count were moderately affected.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases

The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch‐based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon‐optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α‐amylase (SFA1) genes were δ‐integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1‐SFA1] and MEL2[TLG1‐SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch‐to‐ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre‐treatment or commercial enzyme addition) using industrial yeast strains co‐secreting both a glucoamylase and α‐amylase. Biotechnol. Bioeng. 2015;112: 1751–1760.

Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium

The production of bacteriocins from cheap substrates could be useful for many food industrial applications. This study aimed at determining the conditions needed for optimal production of enterocins SD1, SD2, SD3 and SD4 secreted by Enterococcus faecium strains SD1, SD2, SD3 and SD4, respectively. To our knowledge, this is the first use of cheese whey—a low-cost milk by-product—as a substrate for bacteriocin production by E. faecium; skimmed milk and MRS broths were used as reference media. This cheese manufacturing residue proved to be a promising substrate for the production of bacteriocins. However, the levels of secreted antimicrobial compounds were lower than those achieved by E. faecium strains in MRS broth. Bacteriocin production was affected strongly by physical and chemical factors such as growth temperature, time of incubation, pH, and the chemical composition of the culture medium. The optimal temperature and time of incubation supporting the highest bacteriocin production was determined for each strain. Different types, sources and amounts of organic nitrogen, sugar, and inorganic salts played an essential role in bacteriocin secretion. E. faecium strains SD1 and SD2—producing high bacteriocin levels both in cheese whey and skimmed milk—could be of great interest for potential applications in cheese-making.