Carmen Tejedor

The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds

In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S–23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium–Vicia symbiosis.

Assessment of toxicity of river water and effluents by the bioluminescence assay using Photobacterium phosphoreum

Abstract The toxicity bioassay employing Photobacterium phosphoreum (LUMISTOX system) was used to evaluate the evolution of the quality of the River Tormes water during its passage through the city of Salamanca. To do so the toxicity of all the discharges into the river Tormes (22 total) were studied in the stretch in question, together with the impact of such discharges on the river course. Since pollution at low concentrations or the toxicity due to compounds that are strongly diluted in a fast flowing river may go undetected by the system, a technique of organic microcontaminant concentration using XAD-2 resins was used to detect different degrees of toxicity along the stretch studied. To a greater or lesser extent, all the discharges analyzed proved to be toxic for the photobacteria; the impact to the river caused by each was found to be a function of its EC50 and flow rate (Equitox/m 3 ). The toxicity of the water of the river increases (the inhibition of luminescence (%H15), the dilution factor that inhibits luminescence by 20% (G120) and the concentration values leading to a 50% decrease in the emission of light (EC50) for an incubation time of 15 min, increase) as the river receives the different discharges, from the first sample (before the discharges studied), where the water concentrate has a low effective concentration 50 value (EC50 = 1.3) until the last sample (after the discharges studied), where the effective concentration 50 value is maximum (EC50 = 2.9).

Effect of different factors on the die-off of fecal bacteria in a stabilization pond purification plant

A study was made of the effect of aeration on the die-off of different bacterial indicators of fecal waste at the wastewater lagoon purification plant of La Velles (Salamanca, Spain). In situ, the efficacy of the purification system, when the lagoons of the plant were partially aerated, rose from 92, 91, 95 and 17% in the die-off of total coliforms, fecal coliforms, fecal streptococci and sulfite-reducing clostridia to 99, 99, 97 and 53%, respectively. In order to study the effects of the microbial load on the die-off of fecal coliform bacteria and fecal streptococci, in vitro experiments were conducted. The results show that the reduction in the bacterial flora of the wastewater due to different treatments involved a proportional reduction in the die-off of fecal bacteria.

Paenibacillus endophyticus sp. nov., isolated from nodules of Cicer arietinum.

A bacterial strain, designated PECAE04(T), was isolated from root nodules of Cicer arietinum in Spain. Phylogenetic analysis based on 16S rRNA gene sequence placed the isolate into the genus Paenibacillus with its closest relative being Paenibacillus castaneae Ch-32(T) with 98.4 % 16S rRNA gene sequence similarity followed by Paenibacillus glycanilyticus DS-1(T), Paenibacillus prosopidis PW21(T), Paenibacillus xinjiangensis B538(T) and Paenibacillus catalpae D75(T) with similarities ranging from 97.9 to 96.8 %. DNA-DNA hybridization measurements showed values lower than 20 % between the strain PECAE04(T) and any of these species. The isolate was a Gram-stain-positive, motile, sporulating rod. Catalase and oxidase activities were positive. Aesculin was hydrolysed but casein and gelatin were not. Acetoin production, H2S production, nitrate reduction and urease and caseinase production were negative. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the predominant menaquinone and anteiso-C15 : 0, iso-C16 : 0 and C16 : 0 were the major fatty acids. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, a glycolipid, three phospholipids and an unidentified lipid. Meso-diaminopimelic acid was not detected in the peptidoglycan. The DNA G+C content was 52.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain PECAE04(T) should be considered to be a representative of a novel species of the genus Paenibacillus, for which the name Paenibacillus endophyticus sp. nov. is proposed. The type strain is PECAE04(T) ( = LMG 27297(T) = CECT 8234(T)).

Paenibacillus lupini sp. nov., isolated from nodules of Lupinus albus.

A bacterial strain designated RLAHU15(T) was isolated from root nodules of Lupinus albus in Spain. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate in the genus Paenibacillus, with its closest relatives being Paenibacillus catalpae D75(T), Paenibacillus glycanilyticus DS-1(T), Paenibacillus endophyticus PECAE04(T) and Paenibacillus xinjiangensis B538(T) with 98.8 %, 98.9 %, 97.4 % and 97.4 % similarity, respectively. DNA-DNA hybridization studies showed values lower than 45 % between the strain RLAHU15(T) and any of these species. The isolate was a Gram-stain positive, motile and sporulating rod. Catalase activity was weak and oxidase activity was positive. Casein and starch were hydrolysed but gelatin was not. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected and anteiso-C15 : 0 and iso-C16 : 0 were the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids and an unidentified lipid. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 54.4 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RLAHU15(T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillus lupini sp. nov. is proposed. The type strain is RLAHU15(T) ( = LMG 27296(T) = CECT 8235(T)).

Differences in the outer membrane-related properties of the six classical Brucella species

Outer membrane-related properties (such as auto-agglutination and susceptibility to various compounds) of strains representative of the six classical species of the genus Brucella were assessed. The differences identified could not be fully explained based on the smooth or rough phenotype of the strain. Smooth strains of the closely related species Brucella melitensis and B. abortus exhibited different susceptibility patterns and the rough, virulent B. ovis and B. canis strains were equally or more resistant to conditions such as pH, non-immune serum, hydrogen peroxide and bactericidal cationic peptides than smooth strains. Such heterogeneity in outer membrane characteristics could account for differences in pathogenicity and host tropism.

Endobacter medicaginis gen. nov., sp. nov., isolated from alfalfa nodules in an acidic soil

A bacterial strain designated M1MS02(T) was isolated from a surface-sterilized nodule of Medicago sativa in Zamora (Spain). The 16S rRNA gene sequence of this strain showed 96.5 and 96.2 % similarity, respectively, with respect to Gluconacetobacter liquefaciens IFO 12388(T) and Granulibacter bethesdensis CGDNIH1(T) from the family Acetobacteraceae. The novel isolate was a Gram-stain-negative, non-sporulating, aerobic coccoid to rod-shaped bacterium that was motile by a subpolar flagellum. The major fatty acid was C18 : 1ω7c and the major ubiquinone was Q-10. The lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids, three aminolipids, four glycolipids, two phospholipids and one lipid. Strain M1MS02(T) was catalase-positive and oxidase- and urease-negative. Acetate and lactate were not oxidized. Acetic acid was produced from ethanol in culture media supplemented with 2 % CaCO3. Ammonium sulphate was assimilated in glucose medium. The strain produced dihydroxyacetone from glycerol. Phylogenetic and phenotypic analyses commonly used to differentiate genera within the family Acetobacteraceae showed that strain M1MS02(T) should be classified as representing a novel species of a new genus within this family, for which the name Endobacter medicaginis gen. nov., sp. nov. is proposed. The type strain of the type species is M1MS02(T) ( = LMG 26838(T) = CECT 8088(T)). To our knowledge, this is the first report of a member of the Acetobacteraceae occurring as a legume nodule endophyte.

Cohnella lupini sp. nov., an endophytic bacterium isolated from root nodules of Lupinus albus.

A bacterial strain designated RLAHU4B(T) was isolated from root nodules of Lupinus albus in León (Spain). The 16S rRNA gene sequence of this strain showed similarities lower than 97 % with respect to species of the genus Cohnella. The strain was a Gram-variable, sporulating rod, motile by means of peritrichous flagella, and facultatively anaerobic. It was positive for oxidase, catalase and β-galactosidase production but negative for urease, amylase and gelatinase. Strain RLAHU4B(T) grew in the presence of 5 % NaCl. MK-7 was the predominant menaquinone and meso-diaminopimelic acid was present in the peptidoglycan. anteiso-C15 : 0, iso-C16 : 0, iso-C15 : 0 and C16 : 0 were the major fatty acids. Major polar lipids of strain RLAHU4B(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids, two unknown aminophospholipids and one unknown lipid. The DNA G+C content was 57.8 mol%. Strain RLAHU4B(T) presented phenotypic differences from all recognized species of the genus Cohnella. The phylogenetic, chemotaxonomic and phenotypic data indicated that strain RLAHU4B(T) belongs to a novel species of the genus Cohnella, for which the name Cohnella lupini sp. nov. is proposed, with strain RLAHU4B(T) ( = LMG 27416(T) = CECT 8236(T)) as the type strain.

Analysis of the occurrence and distribution of the Omp25/Omp31 family of surface proteins in the six classical Brucella species

Members of the Omp25/Omp31 family of surface proteins were previously shown to participate in the virulence of some Brucella species and a different distribution of the seven proteins of this family among species could be related to the difference in pathogenicity and host preference they exhibit. Accordingly, in this work we have analyzed the expression of the genes coding for the Omp25/Omp31 family in the six classical Brucella species and a set of B. ovis mutant strains with each omp gene inactivated. Immunoblot of whole-cell lysates with antibodies raised against the purified recombinant outer membrane proteins (OMPs) did not show the simultaneous presence of the seven OMPs in any of the Brucella strains studied, but different Omp25/Omp31 profiles were detected, in our experimental conditions, between the Brucella strains representative of the six classical species. Transcripts for omp31, omp25 and omp25c were, in general, the most abundant of the family and some hits were found in B. ovis for a posttranscriptional regulation mechanism and for a compensatory mechanism increasing the synthesis of a protein to compensate for the absence of another one. Finally, the potential interest of Omp25c and Omp31b as subcellular vaccines, considering their occurrence in the Brucella strains studied and their antigenic relatedness with other proteins of the family, is discussed.

Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection.

Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25 d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG1, IgG2a and IgG2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.