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1.  Are drought-resistance promoting bacteria cross-compatible with different plant models? 
Plant Signaling & Behavior  2013;8(10):e26741.
The association between plant and plant growth promoting bacteria (PGPB) contributes to the successful thriving of plants in extreme environments featured by water shortage. We have recently shown that, with respect to the non-cultivated desert soil, the rhizosphere of pepper plants cultivated under desert farming hosts PGPB communities that are endowed with a large portfolio of PGP traits. Pepper plants exposed to bacterial isolates from plants cultivated under desert farming exhibited a higher tolerance to water shortage, compared with untreated control. This promotion was mediated by a larger root system (up to 40%), stimulated by the bacteria, that enhanced plant ability to uptake water from dry soil. We provide initial evidence that the nature of the interaction can have a limited level of specificity and that PGPB isolates may determine resistance to water stress in plants others than the one of the original isolation. It is apparent that, in relation to plant resistance to water stress, a feature of primary evolutionary importance for all plants, a cross-compatibility between PGPB and different plant models exists at least on a short-term.
PMCID: PMC4091069  PMID: 24270625
arid ecosystem; drought tolerance; endosphere; plant growth promoting bacteria; plant-bacteria cross-compatibility; rhizosphere; water stress
2.  Plant Growth Promotion Potential Is Equally Represented in Diverse Grapevine Root-Associated Bacterial Communities from Different Biopedoclimatic Environments 
BioMed Research International  2013;2013:491091.
Plant-associated bacteria provide important services to host plants. Environmental factors such as cultivar type and pedoclimatic conditions contribute to shape their diversity. However, whether these environmental factors may influence the plant growth promoting (PGP) potential of the root-associated bacteria is not widely understood. To address this issue, the diversity and PGP potential of the bacterial assemblage associated with the grapevine root system of different cultivars in three Mediterranean environments along a macrotransect identifying an aridity gradient were assessed by culture-dependent and independent approaches. According to 16S rRNA gene PCR-DGGE, the structure of endosphere and rhizosphere bacterial communities was highly diverse (P = 0.03) and was associated with a cultivar/latitudinal/climatic effect. Despite being diverse, the bacterial communities associated with Egyptian grapevines shared a higher similarity with the Tunisian grapevines than those cultivated in North Italy. A similar distribution, according to the cultivar/latitude/aridity gradients, was observed for the cultivable bacteria. Many isolates (23%) presented in vitro multiple stress resistance capabilities and PGP activities, the most frequent being auxin synthesis (82%), insoluble phosphate solubilisation (61%), and ammonia production (70%). The comparable numbers and types of potential PGP traits among the three different environmental settings indicate a strong functional homeostasis of beneficial bacteria associated with grape root.
PMCID: PMC3708380  PMID: 23878810
3.  Potential for Plant Growth Promotion of Rhizobacteria Associated with Salicornia Growing in Tunisian Hypersaline Soils 
BioMed Research International  2013;2013:248078.
Soil salinity and drought are among the environmental stresses that most severely affect plant growth and production around the world. In this study the rhizospheres of Salicornia plants and bulk soils were collected from Sebkhet and Chott hypersaline ecosystems in Tunisia. Depiction of bacterial microbiome composition by Denaturing Gradient Gel Electrophoresis unveiled the occurrence of a high bacterial diversity associated with Salicornia root system. A large collection of 475 halophilic and halotolerant bacteria was established from Salicornia rhizosphere and the surrounding bulk soil, and the bacteria were characterized for the resistance to temperature, osmotic and saline stresses, and plant growth promotion (PGP) features. Twenty Halomonas strains showed resistance to a wide set of abiotic stresses and were able to perform different PGP activities in vitro at 5% NaCl, including ammonia and indole-3-acetic acid production, phosphate solubilisation, and potential nitrogen fixation. By using a gfp-labelled strain it was possible to demonstrate that Halomonas is capable of successfully colonising Salicornia roots in the laboratory conditions. Our results indicated that the culturable halophilic/halotolerant bacteria inhabiting salty and arid ecosystems have a potential to contribute to promoting plant growth under the harsh salinity and drought conditions. These halophilic/halotolerant strains could be exploited in biofertilizer formulates to sustain crop production in degraded and arid lands.
PMCID: PMC3679824  PMID: 23781499
4.  A Drought Resistance-Promoting Microbiome Is Selected by Root System under Desert Farming 
PLoS ONE  2012;7(10):e48479.
Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of “reverse desertification”. Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress.
Methodology/Principal Findings
We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (Capsicum annuum L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. Bacillus spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by Klebsiella spp. (61% and 44% respectively). Most of the isolates (95%) presented in vitro multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for Bacillus and the rhizobacteria strains. We show that the C. annuum rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress.
Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a “resource island” able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.
PMCID: PMC3485337  PMID: 23119032
5.  Fluorescent-BOX-PCR for resolving bacterial genetic diversity, endemism and biogeography 
BMC Microbiology  2008;8:220.
BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) is one of the most used techniques in biogeography studies of microbial isolates. However the traditional separation of BOX-PCR patterns by agarose gel electrophoresis suffers many limitations. The aim of this research was to set up a fluorescent BOX-PCR (F-BOX-PCR) assay in which separation of PCR products is automated in a capillary electrophoresis system. F-BOX-PCR was compared with the traditional BOX-PCR using bacterial strains with different G+C content (Bacillus cereus; Escherichia coli; isolates of the family Geodermatophilaceae). Resolution, discriminatory power and reproducibility were evaluated by assaying different electrophoretic runs, PCR reactions and independent DNA extractions. BOX-PCR and F-BOX-PCR were compared for the analysis of 29 strains of Modestobacter multiseptatus isolated from three different microsites in an altered carbonatic wall from Cagliari, Italy, and 45 strains of Streptococcus thermophilus isolated from 34 samples of the hand-made, yogurt-like product Matsoni, collected in different locations in Georgia.
Fluorophore 6-FAM proved more informative than HEX and BOX-PCR both in agarose gel electrophoresis (p < 0.004 and p < 0.00003) and in capillary electrophoresis (compared only with HEX, p < 2 × 10-7). 6-FAM- and HEX-based F-BOX-PCR respectively detected up to 12.0 and 11.3 times more fragments than BOX-PCR. Replicate separations of F-BOX-PCR showed an accuracy of the size calling of ± 0.5 bp until 500 bp, constantly decreasing to ± 10 bp at 2000 bp. Cluster analysis of F-BOX-PCR profiles grouped M. multiseptatus strains according to the microsite of isolation and S. thermophilus strains according to the geographical origin of Matsoni, but resulted intermixed when a BOX-PCR dataset was used.
F-BOX-PCR represents an improved method for addressing bacterial biogeography studies both in term of sensitivity, reproducibility and data analysis.
PMCID: PMC2625358  PMID: 19077307
6.  DNA is preserved and maintains transforming potential after contact with brines of the deep anoxic hypersaline lakes of the Eastern Mediterranean Sea 
Saline Systems  2008;4:10.
Extracellular dissolved DNA has been demonstrated to be present in many terrestrial and aquatic environments, actively secreted, or released by decaying cells. Free DNA has the genetic potential to be acquired by living competent cells by horizontal gene transfer mediated by natural transformation. The aim of this work is to study the persistence of extracellular DNA and its biological transforming activity in extreme environments like the deep hypersaline anoxic lakes of the Mediterranean Sea. The brine lakes are separated from the upper seawater by a steep chemocline inhabited by stratified prokaryotic networks, where cells sinking through the depth profile encounter increasing salinity values and osmotic stress.
Seven strains belonging to different taxonomic groups isolated from the seawater-brine interface of four hypersaline lakes were grown at medium salinity and then incubated in the brines. The osmotic stress induced the death of all the inoculated cells in variable time periods, between 2 hours and 144 days, depending on the type of brine rather than the taxonomic group of the strains, i.e. Bacillaceae or gamma-proteobacteria. The Discovery lake confirmed to be the most aggressive environment toward living cells. In all the brines and in deep seawater dissolved plasmid DNA was substantially preserved for a period of 32 days in axenic conditions. L'Atalante and Bannock brines induced a decrease of the supercoiled form up to 70 and 40% respectively; in the other brines only minor changes in plasmid conformation were observed. Plasmid DNA after incubation in the brines maintained the capacity to transform naturally competent cells of Acinetobacter baylii strain BD413.
Free dissolved DNA is likely to be released by the lysis of cells induced by osmotic stress in the deep hypersaline anoxic lakes. Naked DNA was demonstrated to be preserved and biologically active in these extreme environments, and hence could constitute a genetic reservoir of traits acquirable by horizontal gene transfer.
PMCID: PMC2531117  PMID: 18681968
7.  Strategy for In Situ Detection of Natural Transformation-Based Horizontal Gene Transfer Events▿ †  
A strategy is described that enables the in situ detection of natural transformation in Acinetobacter baylyi BD413 by the expression of a green fluorescent protein. Microscale detection of bacterial transformants growing on plant tissues was shown by fluorescence microscopy and indicated that cultivation-based selection of transformants on antibiotic-containing agar plates underestimates transformation frequencies.
PMCID: PMC2258602  PMID: 18165369
8.  A Novel Reductive Dehalogenase, Identified in a Contaminated Groundwater Enrichment Culture and in Desulfitobacterium dichloroeliminans Strain DCA1, Is Linked to Dehalogenation of 1,2-Dichloroethane▿  
A mixed culture dechlorinating 1,2-dichloroethane (1,2-DCA) to ethene was enriched from groundwater that had been subjected to long-term contamination. In the metagenome of the enrichment, a 7-kb reductive dehalogenase (RD) gene cluster sequence was detected by inverse and direct PCR. The RD gene cluster had four open reading frames (ORF) showing 99% nucleotide identity with pceB, pceC, pceT, and orf1 of Dehalobacter restrictus strain DSMZ 9455T, a bacterium able to dechlorinate chlorinated ethenes. However, dcaA, the ORF encoding the catalytic subunit, showed only 94% nucleotide and 90% amino acid identity with pceA of strain DSMZ 9455T. Fifty-three percent of the amino acid differences were localized in two defined regions of the predicted protein. Exposure of the culture to 1,2-DCA and lactate increased the dcaA gene copy number by 2 log units, and under these conditions the dcaA and dcaB genes were actively transcribed. A very similar RD gene cluster with 98% identity in the dcaA gene sequence was identified in Desulfitobacterium dichloroeliminans strain DCA1, the only known isolate that selectively dechlorinates 1,2-DCA but not chlorinated ethenes. The dcaA gene of strain DCA1 possesses the same amino acid motifs as the new dcaA gene. Southern hybridization using total genomic DNA of strain DCA1 with dcaA gene-specific and dcaB- and pceB-targeting probes indicated the presence of two identical or highly similar dehalogenase gene clusters. In conclusion, these data suggest that the newly described RDs are specifically adapted to 1,2-DCA dechlorination.
PMCID: PMC1892866  PMID: 17351102
9.  Synthetic Consolidants Attacked by Melanin-Producing Fungi: Case Study of the Biodeterioration of Milan (Italy) Cathedral Marble Treated with Acrylics▿  
Monuments and artistic stone surfaces are often consolidated and protected with synthetic polymers, in particular, acrylics. Although it is generally thought that acrylic polymers are resistant to biodeterioration, we report for the first time the systematic occurrence of dematiaceous meristematic fungi on many marble samples of the cathedral in Milan (Italy) previously treated with this material. Fourier transform infrared spectroscopy applied to the Milan cathedral stone samples revealed characteristic features of biodeteriorated synthetic resins that differentiated them from the aged but nonbiodeteriorated samples. Samples showing biological colonization were analyzed for the presence of fungi. Cultivation and morphological characterization and methods independent from cultivation, such as denaturing gradient gel electrophoresis coupled with partial 18S rRNA gene sequencing and immunofluorescence staining with melanin-binding antibodies, showed that melanin-producing species are heavily present on stone surfaces protected with acrylic resins. This observation raises the question of the effectiveness of acrylics in protecting stone artworks.
PMCID: PMC1797126  PMID: 17071788
10.  Diversity of Bacillus anthracis Strains in Georgia and of Vaccine Strains from the Former Soviet Union 
Despite the increased number of anthrax outbreaks in Georgia and the other Caucasian republics of the former Soviet Union, no data are available on the diversity of the Bacillus anthracis strains involved. There is also little data available on strains from the former Soviet Union, including the strains previously used for vaccine preparation. In this study we used eight-locus variable-number tandem repeat analyses to genotype 18 strains isolated from infected animals and humans at different sites across Georgia, where anthrax outbreaks have occurred in the last 10 years, and 5 strains widely used for preparation of human and veterinary vaccines in the former Soviet Union. Three different genotypes affiliated with the A3.a cluster were detected for the Georgian isolates. Two genotypes were previously shown to include Turkish isolates, indicating that there is a regional strain pattern in the South Caucasian-Turkish region. Four of the vaccine strains were polymorphic, exhibiting three different patterns of the cluster A1.a genotype and the cluster A3.b genotype. The genotype of vaccine strain 71/12, which is considered an attenuated strain in spite of the presence of both of the virulence pXO plasmids, appeared to be a novel genotype in the A1.a cluster.
PMCID: PMC1538727  PMID: 16885320
11.  Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis†  
Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.
PMCID: PMC1392923  PMID: 16461679
12.  Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities 
Applied and Environmental Microbiology  2004;70(10):6147-6156.
ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the façade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng μl−1, while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.
PMCID: PMC522057  PMID: 15466561
13.  Nature of Polymorphisms in 16S-23S rRNA Gene Intergenic Transcribed Spacer Fingerprinting of Bacillus and Related Genera 
The intergenic transcribed spacers (ITS) between the 16S and 23S rRNA genetic loci are frequently used in PCR fingerprinting to discriminate bacterial strains at the species and intraspecies levels. We investigated the molecular nature of polymorphisms in ITS-PCR fingerprinting of low-G+C-content spore-forming bacteria belonging to the genera Bacillus, Brevibacillus, Geobacillus, and Paenibacillus. We found that besides the polymorphisms in the homoduplex fragments amplified by PCR, heteroduplex products formed during PCR between amplicons from different ribosomal operons, with or without tRNA genes in the ITS, contribute to the interstrain variability in ITS-PCR fingerprinting patterns obtained in polyacrylamide-based gel matrices. The heteroduplex nature of the discriminating bands was demonstrated by fragment separation in denaturing polyacrylamide gels, by capillary electrophoresis, and by cloning, sequencing, and recombination of purified short and tRNA gene-containing long ITS. We also found that heteroduplex product formation is enhanced by increasing the number of PCR cycles. Homoduplex-heteroduplex polymorphisms (HHP) in a conserved region, such as the 16S and 23S rRNA gene ITS, allowed discrimination of closely related strains and species undistinguishable by other methods, indicating that ITS-HHP analysis is an easy and reproducible additional tool for strain typing.
PMCID: PMC194986  PMID: 12957895
14.  Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes 
Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified from several strains of the six species of the Bacillus cereus group showed that B. anthracis Davis TE702 and B. mycoides G2 have other intermediate fragments in addition to the 220- and 550-bp homoduplex fragments typical of the B. cereus group. Long and intermediate homoduplex ITS fragments from strains Davis TE702 and G2 and from another 19 strains of the six species were sequenced. Two main types of ITS were found, either with two tRNA genes (tRNAIle and tRNAAla) or without any at all. Strain Davis TE702 harbors an additional ITS with a single tRNA gene, a hybrid between the tRNAIle and tRNAAla genes, suggesting that a recombination event rather than a deletion generated the single tDNA-containing ITS. Strain G2 showed an additional ITS of intermediate length with no tDNA and no similarity to other known sequences. Neighbor-joining analysis of tDNA-containing long ITS indicated that B. cereus and B. thuringiensis represent a single clade. Three signature sequences discriminated B. anthracis from B. cereus and B. thuringiensis, indicating that the anthrax agent started evolving separately from the related clades of the B. cereus group. B. mycoides and B. weienstephanensis were very closely related, while B. pseudomycoides appeared the most distant species.
PMCID: PMC152393  PMID: 12513974
15.  Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “Bacillus cereus Group” 
Applied and Environmental Microbiology  2000;66(12):5460-5468.
Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the “B. cereus group,” advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.
PMCID: PMC92482  PMID: 11097928
16.  A Randomly Amplified Polymorphic DNA Marker Specific for the Bacillus cereus Group Is Diagnostic for Bacillus anthracis 
Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group.
PMCID: PMC91177  PMID: 10049896

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