Agrobacterium leguminum sp. nov., isolated from nodules of Phaseolus vulgaris in Spain.

Two endophytic strains, coded MOVP5T and MOPV6, were isolated from nodules of Phaseolus vulgaris plants grown on agricultural soil in Southeastern Spain, and were characterized through a polyphasic taxonomy approach. Their 16S rRNA gene sequences showed 99.3 and 99.4 %, 98.9 and 99.6 %, and 99.0 and 98.7% similarity to 'A. deltaense' YIC 4121T, A. radiobacter LGM 140T, and A. pusense NRCPB10T, respectively. Multilocus sequence analysis based on sequences of recA and atpD genes suggested that these two strains could represent a new Agrobacterium species with less than 96.5 % similarity to their closest relatives. PCR amplification of the telA gene, involved in synthesis of protelomerase, confirmed the affiliation of strains MOPV5T and MOPV6 to the genus Agrobacterium. Whole genome average nucleotide identity and digital DNA-DNA hybridization average values were less than 95.1 and 66.7 %, respectively, with respect to its closest related species. Major fatty acids in strain MOPV5T were C18 : 1 ω7c/C18 : 1 ω6c in summed feature 8, C19 : 0 cyclo ω8c, C16 : 0 and C16 : 0 3-OH. Colonies were small to medium, pearl-white coloured on YMA at 28 °C and growth was observed at 10-42 °C, pH 5.0-10.0 and with 0.0-0.5 % (w/v) NaCl. The DNA G+C content was 59.9 mol%. These two strains differ from all other genomovars of Agrobacterium found so far, including those that have not yet given a Latin name. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strain MOPV5T as representing a novel species of Agrobacterium, for which the name Agrobacterium leguminum sp. nov. is proposed. The type strain is MOPV5T (=CECT 30096T=LMG 31779T).

Agrobacterium cavarae sp. nov., isolated from maize (Zea mays L.) roots.

A bacterial strain designated as RZME10T was isolated from a Zea mays L. root collected in Spain. Results of analysis of the 16S rRNA gene sequence showed that this strain belongs to the genus Agrobacterium with Agrobacterium larrymoorei ATCC 51759T being the most closely related species with 99.9 % sequence similarity. The similarity values of the rpoB, recA, gyrB, atpD and glnII genes between strain RZME10T and A. larrymoorei ATCC 51759T were 93.5, 90.0, 88.7, 87.9 and 90.1 %, respectively. The estimated average nucleotide identity using blast and digital DNA-DNA hybridization values between these two strains were 80.4 and 30.2 %, respectively. The major fatty acids of strain RZME10T are those from summed feature 8 (C18 : 1 ω6c/C18 : 1 ω7c) and C16 : 0. Pathogenicity tests on tomato and carrot roots showed that strain RZME10T was not able to induce plant tumours. Based on the results of genomic, chemotaxonomic and phenotypic analyses, we propose that strain RZME10T represents a novel species named Agrobacterium cavarae sp. nov. (type strain RZME10T=CECT 9795T=LMG 31257T).

Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L.

Four strains, coded as UPM1132, UPM1133T, UPM1134 and UPM1135, and isolated from nodules of Pisum sativum plants grown on Ni-rich soils were characterised through a polyphasic taxonomy approach. Their 16S rRNA gene sequences were identical and showed 100% similarity with their closest phylogenetic neighbors, the species included in the 'R. leguminosarum group': R. laguerreae FB206T, R. leguminosarum USDA 2370T, R. anhuiense CCBAU 23252T, R. sophoreae CCBAU 03386T, R. acidisoli FH13T and R. hidalgonense FH14T, and 99.6% sequence similarity with R. esperanzae CNPSo 668T. The analysis of combined housekeeping genes recA, atpD and glnII sequences showed similarities of 92-95% with the closest relatives. Whole genome average nucleotide identity (ANI) values were 97.5-99.7% ANIb similarity among the four strains, and less than 92.4% with closely related species, while digital DNA-DNA hybridization average values (dDDH) were 82-85% within our strains and 34-52% with closely related species. Major fatty acids in strain UPM1133T were C18:1 ω7c / C18:1 ω6c in summed feature 8, C14:0 3OH/ C16:1 iso I in summed feature 2 and C18:0. Colonies were small to medium, pearl-white coloured in YMA at 28°C and growth was observed in the ranges 8-34°C, pH 5.5-7.5 and 0-0.7% (w/v) NaCl. The DNA G+C content was 60.8mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains UPM1132, UPM1133T, UPM1134 and UPM1135 into a novel species of Rhizobium, for which the name Rhizobium ruizarguesonis sp. nov. is proposed. The type strain is UPM1133T (=CECT 9542T=LMG 30526T).

History and current taxonomic status of genus Agrobacterium.

The genus Agrobacterium was created a century ago by Conn who included it in the family Rhizobiaceae together with the genus Rhizobium. Initially, the genus Agrobacterium contained the non-pathogenic species Agrobacterium radiobacter and the plant pathogenic species Agrobacterium tumefaciens and Agrobacterium rhizogenes. At the end of the past century two new pathogenic species, Agrobacterium rubi and Agrobacterium vitis, were added to the genus. Already in the present century these species plus Agrobacterium larrymoorei were reclassified into genus Rhizobium. This reclassification was controversial and for a time both genus names were used when new species were described. Few years ago, after a taxonomic revision based on genomic data, the old species A. rhizogenes was maintained in the genus Rhizobium, the old species A. vitis was transferred to the genus Allorhizobium and several Rhizobium species were transferred to the genus Agrobacterium, which currently contains 14 species including the old species A. radiobacter, A. tumefaciens, A. rubi and A. larrymoorei. Most of these species are able to produce tumours in different plants, nevertheless the genus Agrobacterium also encompasses non-pathogenic species, one species able to nodulate legumes and one human pathogenic species. Taking into account that the species affiliations to five Agrobacterium genomospecies have not been determined yet, an increase in the number of species within this genus is expected in the near future.

Pseudomonas turukhanskensis sp. nov., isolated from oil-contaminated soils.

A bacterial strain named IB1.1T was isolated in a screening of hydrocarbon-degrading bacteria from oil-contaminated soils on the territory of the Turukhansk District of Krasnoyarsk Krai, East Siberia, Russia. The 16S rRNA gene sequence had 98.7 % identity with respect to the closest phylogenetic relative, Pseudomonas granadensis F-278,770T, and the next most closely related species with 98.6 % similarity was Pseudomonaspunonensis, suggesting that IB1.1T should be classified within the genus Pseudomonas. The analysis of housekeeping genes rpoB, rpoD and gyrB showed similarities lower than 90 % in all cases with respect to the closest relatives, confirming its phylogenetic affiliation. The strain showed a polar flagellum. The respiratory quinone was Q9. The major fatty acids were 16 : 1ω7c/16 : 1ω6c (summed feature 3), 18 : 1ω7c and 16 : 0. The strain was oxidase- and catalase-positive, but the arginine dihydrolase system was not present. Nitrate reduction, urease and ß-galactosidase production, and aesculin hydrolysis were negative. The temperature range for growth was 4-34 °C, and the strain could grow at pH 11. The DNA G+C content was 58.5 mol%. DNA-DNA hybridization results showed values of less than 30 % relatedness with respect to the type strains of the eight most closely related species. Therefore, the dataset of genotypic, phenotypic and chemotaxonomic data support the classification of strain IB1.1T into a novel species of the genus Pseudomonas, for which the name Pseudomonasturukhanskensis sp. nov. is proposed. The type strain is IB1.1T (=VKM B-2935T=CECT 9091T).

Bradyrhizobium centrosemae (symbiovar centrosemae) sp. nov., Bradyrhizobium americanum (symbiovar phaseolarum) sp. nov. and a new symbiovar (tropici) of Bradyrhizobium viridifuturi establish symbiosis with Centrosema species native to America.

In this work we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Venezuela from root nodules of Centrosema species. The analysis of the 16S rRNA gene showed that the strains belong to three clusters within genus Bradyrhizobium which have 100% similarity with Bradyrhizobium daqingense CCBAU 15774(T)Bradyrhizobium guangxiense CCBAU 53363(T) and Bradyrhizobium viridifuturi SEMIA 690(T). The results of recA and glnII gene analysis confirmed the identification of the strains CMVU02 and CMVU30 as Bradyrhizobium viridifuturi but the nodC gene analysis showed that they belong to a new symbiovar for which we propose the name tropici. Nevertheless, the concatenated recA and glnII gene phylogenetic analysis, DNA-DNA hybridization and phenotypic characterization showed that the strains A9(T), CMVU44(T) and CMVU04 belong to two novel Bradyrhizobium species. The analysis of the nodC gene showed that these strains also represent two new symbiovars. Based on these results we propose the classification of the strain A9(T) isolated from Centrosema molle into the novel species Bradyrhizobium centrosemae (sv. centrosemae) sp. nov. (type strain A9(T)=LMG 29515(T)=CECT 9095(T)). and the classification of the strains CMVU44(T) and CMVU04 isolated from C. macrocarpum into the novel species Bradyrhizobium americanum (sv. phaseolarum) sp. nov. (type strain CMVU44(T)=LMG 29514(T)=CECT 9096(T)).

Massilia violacea sp. nov., isolated from riverbank soil.

A bacterial strain designated CAVIOT was isolated during the course of a study of culturable bacteria in a riverbank soil sample from Tlaxcala, Mexico. The strain was subjected to a polyphasic taxonomic characterization. Strain CAVIOT was aerobic, Gram-stain-negative, non-spore-forming and rod-shaped. Colonies grown on R2A agar at 28 °C were pale violet, mucoid, rounded, smooth and glossy. The strain was motile and catalase- and oxidase-positive, and maximum growth temperature was 35 °C. Strain CAVIOT was classified within the genus Massilia as its 16S rRNA gene sequence was closely related to those of Massilia umbonata LP01T (97.5 % similarity), Massilia dura 16T (97.2 %) and Massilia plicata 76T (97.1 %). The predominant respiratory quinone was Q8. The major fatty acids were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c). The predominant polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and an unknown phospholipid. The DNA G+C content was 65.0 mol% (Tm). DNA-DNA hybridization results showed values below 25 % with respect to the type strains of the closest related species. Therefore, strain CAVIOT can be differentiated from previously described species of the genus Massilia and represents a novel species, for which the name Massilia violacea sp. nov. is proposed. The type strain is CAVIOT ( = CECT 8897T = LMG 28941T).

Erwinia endophytica sp. nov., isolated from potato (Solanum tuberosum L.) stems.

We analysed, using a polyphasic taxonomic approach, two bacterial strains coded BSTT30T and BSTT40, isolated in the course of a study of endophytic bacteria occurring in the stems and roots of potatoes growing in soil from Salamanca, Spain. The 16S rRNA gene sequence was identical in both strains and had 98.4 % identity with respect to the closest relatives Erwinia tasmaniensis Et1/99T and Erwinia rhapontici ATCC29283T. Erwinia billingiae E63T and Erwinia toletana A37T were also closely related with 98.2 % sequence similarities, so the novel strains were classified within the genus Erwinia. The analysis of the housekeeping genes gpd, gyrB and rpoD confirmed the phylogenetic affiliation of strains BSTT30T and BSTT40 with similarities of lower than 90 % in all cases with respect to the closest relatives mentioned above. The respiratory quinone of strain BSTT30T was Q8. The major fatty acids were C16 : 0, C16 : 1ω7c/16 : 1ω6c in summed feature 3 and C18 : 1ω7c/18 : 2ω6,9c in summed feature 8. The novel strains were oxidase-negative and catalase-positive. Glucose was fermented without gas production. They were negative for arginine dihydrolase, urease and indole production. The strains could grow at 35 °C and at pH 10. DNA G+C content was 50.1 mol%. DNA-DNA hybridization results showed values of lower than 29 % relatedness with respect to the type strains of the four most closely related species. Therefore, the combined genotypic, phenotypic and chemotaxonomic data support the classification of strains BSTT30T and BSTT40 into a novel species of the genus Erwinia, for which the name Erwinia endophytica sp. nov. is proposed. The type strain is BSTT30T ( = LMG 28457T, CECT 8692T).

Inoculation of the nonlegume Capsicum annuum (L.) with Rhizobium strains. 1. Effect on bioactive compounds, antioxidant activity, and fruit ripeness.

Pepper (Capsicum annuum L.) is an economically important agricultural crop and an excellent dietary source of natural colors and antioxidant compounds. The levels of these compounds can vary according to agricultural practices, like inoculation with plant growth-promoting rhizobacteria. In this work we evaluated for the first time the effect of the inoculation of two Rhizobium strains on C. annuum metabolites and bioactivity. The results revealed a decrease of organic acids and no effect on phenolics and capsaicinoids of leaves from inoculated plants. In the fruits from inoculated plants organic acids and phenolic compounds decreased, showing that fruits from inoculated plants present a higher ripeness stage than those from uninoculated ones. In general, the inoculation with Rhizobium did not improve the antioxidant activity of pepper fruits and leaves. Considering the positive effect on fruit ripening, the inoculation of C. annuum with Rhizobium is a beneficious agricultural practice for this nonlegume.

Physiological and antioxidant responses of Medicago sativa-rhizobia symbiosis to cyanobacterial toxins (Microcystins) exposure.

Toxic cyanobacteria in freshwaters can induce potent harmful effects on growth and development of plants irrigated with contaminated water. In this study, the effect of cyanobacteria extract containing Microcystins (MC) on Medicago sativa-rhizobia symbiosis was investigated in order to explore plants response through biomass production, photosynthetic pigment and antioxidant enzymes analysis: Peroxidase (POD), Polyphenoloxidase (PPO) and Catalase (CAT). Alfalfa plants were inoculated with two endosymbiotic rhizobial strains: RhOL1 (MC less sensitive strain) and RhOL3 (MC more sensitive strain), to evaluate the rhizobial contribution on the plant response cultured under cyanobacterial toxins stress. The two rhizobia strains were identified as Ensifer meliloti by sequence analysis of their rrs and atpD genes. The chronic exposure to MC extract showed shoot, root and nodules dry weight decrease, in both symbiosis cultures. The rate of decline in plants inoculated with RhOL3 was higher than that in symbiosis with RhOL1 mainly at 20 µg L(-1) of MC. Cyanotoxins also reduced photosynthetic pigment content and generated an oxidative stress observed at cellular level. POD, PPO and CAT activities were significantly increased in leaves, roots and nodules of alfalfa plants exposed to MC. These enzyme activities were higher in plants inoculated with RhOL3 especially when alfalfa plants were exposed to 20 µg L(-1) of MC. The present paper reports new scientific finding related to the behavior of rhizobia-M. sativa associations to MC (Microcystins) for later recommendation concerning the possible use of these symbiosis face to crops exposure to MC contaminated water irrigation.

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma.

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086(T), with 99.41% identity with respect to the strain Ro19(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19(T) and B. lablabi CCBAU 23086(T), with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086(T). Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19(T)=LMG 27393(T)=CECT 8261(T)). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name "retamae", that mainly contains nodulating strains isolated from Retama species in different continents.

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif.

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).

Mesorhizobial strains nodulating Anagyris latifolia and Lotus berthelotii in Tamadaya ravine (Tenerife, Canary Islands) are two symbiovars of the same species, Mesorhizobium tamadayense sp. nov.

Barranco de Tamadaya is a deep ravine located in southern Tenerife, which is included within a protected area where several endemic plants grow. Among them, two legumes are catalogued as critically endangered, Anagyris latifolia and Lotus berthelotii. Rhizobial strains isolated from their root nodules grown in soil samples from this ravine harboured symbiotic genes belonging to two distant symbiovars, but they shared identical 16S rRNA gene sequences (rrs). The phylogeny based on the rrs sequences placed these isolates in a separate subbranch that did not include any of the currently recognised Mesorhizobium species, but the resolution of the ribosomal tree did not permit further taxonomic conclusions. Nevertheless, multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, recA, glnII and dnaK) and the rrs gene generated a highly supported Bayesian phylogeny, identifying these isolates as a new Mesorhizobium lineage. DNA-DNA hybridisation homology percentages were lower than 30% compared to type strains of the closest related species, and supported the phylogenetic data. Phenotypic characterisation also distinguished this lineage from the other closest Mesorhizobium species. The polyphasic approach thus confirmed that the isolates represented a novel species for which we propose the name Mesorhizobium tamadayense sp. nov. The type strain is Ala-3(T) (CECT 8040(T), LMG 26736(T)).

[Microorganisms and cancer: scientific evidence and new hypotheses]. / Microorganismos y cáncer: evidencias científicas y nuevas hipótesis.

Microorganism involvement in cancer has been known for over a century, and different types of parasites, bacteria and viruses have been associated with oncogenic processes. Among the bacteria, the first recognised was Helicobacter pylori which causes gastric cancer and might be related to extra-gastric cancer in humans. Helicobacter hepaticus has been associated with liver cancers using animal models. Other bacteria, such as Chlamydia psitacii, Borrelia burgdorferi and Streptococcus bovis, have been associated with ocular, skin and colorectal cancers, respectively. Also, a commensal bacterium in the human intestine, Bacteroides fragilis, has been linked, very recently, with colorectal cancer using animal models.

Analysis of core genes supports the reclassification of strains Agrobacterium radiobacter K84 and Agrobacterium tumefaciens AKE10 into the species Rhizobium rhizogenes.

Some strains of the former genus Agrobacterium have high biotechnological interest and are currently misclassified. Consequently, in this study, the taxonomic status of the non-pathogenic strain Agrobacterium radiobacter K84, used in biological control, and the tumourigenic strain Agrobacterium tumefaciens AKE10, able to regenerate tobacco transgenic plants, was revised. The phylogenetic analysis of the chromosomal genes rrs, atpD and recA showed that they should be reclassified into Rhizobium rhizogenes. The analysis of virulence genes located in the Ti plasmid (pTi) outside T-DNA showed a common phylogenetic origin among strains AKE10, R. rhizogenes 163C and A. tumefaciens (currently R. radiobacter) C58. However, the genes located inside the T-DNA, mainly the 6b gene, of strain AKE10 were phylogenetically close to those of strain 163C but divergent from those of strain C58. Furthermore, the T-DNA of tumourigenic strains from R. rhizogenes conferred on them the ability to regenerate tumour tissue resembling fasciation in tobacco plants. These results showed the existence of a highly mosaic genetic organization in tumourigenic strains of the genus Rhizobium and provided evidence of the involvement of T-DNA from tumourigenic strains of R. rhizogenes in fasciation of Nicotiana leaves. The data further suggested that pathogenic strains of Rhizobium could be good models to analyse bacterial evolution.

Alcanivorax balearicus sp. nov., isolated from Lake Martel.

A bacterial strain designated MACL04(T) was isolated from Lake Martel, a subterraneous saline lake in Mallorca (Spain). The complete 16S rRNA gene sequence of this strain showed nearly 100 % similarity to that of Alcanivorax dieselolei B-5(T). Despite this high similarity, strain MACL04(T) showed phenotypic, chemotaxonomic and molecular differences with respect to A. dieselolei, indicating that strain MACL04(T) represents a separate species. Cells of strain MACL04(T) were motile by means of a single polar or subpolar flagellum and colonies formed on media containing 1 % (v/v) Tween 20 were opaque and mucoid, with blue-green iridescence. The generation time of strain MACL04(T) in this medium was approximately half that of A. dieselolei B-5(T) and strain MACL04(T) did not produce lipases after incubation for 5 days. Strain MACL04(T) did not require NaCl for growth and grew in the presence of up to 15 % (w/v) NaCl. The strain was able to use alkanes as a sole carbon source; however, glucose could also be used, albeit weakly, as a carbon source. Several amino acids and organic acids were used as carbon sources. Strain MACL04(T) produced acid in media containing pyruvate as the sole carbon source. The major fatty acids were C(19 : 0) cyclo omega8c and C(16 : 0). The fatty acid C(16 : 1)omega8c, present in strain MACL04(T), was not detected in the recognized Alcanivorax species. The sequences of the large and short 16S-23S intergenic spacer regions showed similarities of 97.2 and 98.8 % (ungapped) with respect to A. dieselolei B-5(T). Partial sequences of gyrB and alkb genes showed 94.0 % similarity between strain MACL04(T) and A. dieselolei B-5(T). The G+C content of strain MACL04(T) was 62.8 mol%. The data from this polyphasic study indicate that strain MACL04(T) represents a novel species of the genus Alcanivorax, for which the name Alcanivorax balearicus sp. nov. is proposed. The type strain is MACL04(T) (=LMG 22508(T)=CECT 5683(T)).

Burkholderia ferrariae sp. nov., isolated from an iron ore in Brazil.

A Gram-negative, non-spore-forming bacterial strain with the ability to solubilize highly insoluble phosphatic minerals was isolated from a high-phosphorous iron ore from Minas Gerais State, Brazil. This strain, designated FeGl01(T), was subjected to a polyphasic taxonomic investigation. Comparative 16S rRNA gene sequence analysis indicated that it formed a distinct phylogenetic lineage within the genus Burkholderia together with several other species of the genus, e.g. Burkholderia sacchari, Burkholderia tropica and Burkholderia unamae. Partial nucleotide sequencing and analysis of the recA gene roughly corroborated the phylogenetic position of strain FeGl01(T) within the genus Burkholderia. The chemotaxonomic properties of strain FeGl01(T), such as ubiquinone Q-8 as the predominant quinone system and C(16 : 0), C(17 : 0) cyclo, C(18 : 1)omega7c and C(19 : 0)omega8c cyclo as the major fatty acids, were also consistent with its classification within the genus Burkholderia. DNA-DNA hybridization experiments between strain FeGl01(T) and the type strains of B. unamae, B. sacchari and B. tropica yielded reassociation values of 40 % or lower, which, together with qualitative and quantitative differences in fatty acid composition and with differences in several phenotypic traits, support the separation of the new isolate from the phylogenetically most closely related species. Therefore, it is suggested that strain FeGl01(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia ferrariae sp. nov. is proposed. The type strain is FeGl01(T) (=LMG 23612(T)=CECT 7171(T)=DSM 18251(T)).

Application of horizontal staircase electrophoresis in agarose minigels to the random intergenic spacer analysis of clinical samples.

The random intergenic spacer analysis is a recently developed technique for the study of microbial populations. The bacterial intergenic spacer (ITS) is located between 16S rRNA and 23S rRNA genes and presents different length and sequence among bacterial species. Therefore, the amplicons can be separated by electrophoresis commonly performed at low voltage during several hours. Although this technique is especially useful for unculturable microorganisms, it has not been applied before to clinical sample analysis. As these samples have a limited number of bacterial species, the size of the gels may be reduced to facilitate their handling and to reduce the running time. To obtain maximum separation among the ITS bands, we analysed in this work different electrophoretical conditions including staircase electrophoresis, a technique based on the application of several voltage steps. The results obtained showed a different behaviour of the electrical resistance during the performance of submarine horizontal and vertical staircase electrophoresis. In the first case the resistance decreased during most of the running time whereas in the second case it increased. Here, we show that the performance of horizontal staircase electrophoresis reduces the running time more than 80% with respect to conventional electrophoresis at low voltages. This procedure was applied to the separation of ITS bands from bacterial DNA present in a tissue from a vocal cord biopsy. The sequencing of these bands allowed their identification. This new procedure may be very useful in the rapid diagnosis of bacteria present in human, animal and plant tissues.

Pseudomonas argentinensis sp. nov., a novel yellow pigment-producing bacterial species, isolated from rhizospheric soil in Cordoba, Argentina.

During a study in the Argentinian region of Chaco (Cordoba), some strains were isolated from the rhizosphere of grasses growing in semi-desertic arid soils. Two of these strains, one isolated from the rhizospheric soil of Chloris ciliata (strain CH01(T)) and the other from Pappophorum caespitosum (strain PA01), were Gram-negative, strictly aerobic rods, which formed yellow round colonies on nutrient agar. They produced a water-insoluble yellow pigment, and a fluorescent pigment was also detected. A polyphasic taxonomic approach was used to characterize the strains. Comparison of the 16S rRNA gene sequences showed a similarity of 99.3 % between them, and phylogenetic analysis revealed that the strains belong to the genus Pseudomonas, within the gamma-subclass of the Proteobacteria. The closest related species is Pseudomonas straminea IAM 1598(T) (similarity of 99.0 % to strain CH01(T) and 98.8 % to strain PA01), clustering in a separate branch with the various methods of tree building used. Strains CH01(T) and PA01 both had a single polar flagellum, like other yellow pigment-producing pseudomonads related to them. Both strains produced catalase and oxidase. Similar to P. straminea, they did not hydrolyse gelatin or casein. The G+C DNA contents determined were 57.5 mol% for CH01(T) and 58.0 mol% for PA01. DNA-DNA hybridization results showed 81 % relatedness between them, and only 40-44 % relatedness with respect to the type strain of P. straminea. These results, together with other phenotypic characteristics, support the conclusion that both isolates belong to the same species, and should be described as representing a novel species within the genus Pseudomonas, for which the name Pseudomonas argentinensis sp. nov. is proposed. The type strain is CH01(T) (=LMG 22563(T) = CECT 7010(T)).

Reclassification of Agrobacterium ferrugineum LMG 128 as Hoeflea marina gen. nov., sp. nov.

Members of the species Agrobacterium ferrugineum were isolated from marine environments. The type strain of this species (= LMG 22047(T) = ATCC 25652(T)) was recently reclassified in the new genus Pseudorhodobacter, in the order 'Rhodobacterales' of the class 'Alphaproteobacteria'. Strain LMG 128 (= ATCC 25654) was also initially classified as belonging to the species Agrobacterium ferrugineum; however, the nearly complete 16S rRNA gene sequence of this strain indicated that it does not belong within the genus Agrobacterium or within the genus Pseudorhodobacter. The closest related organism, with 95.5 % 16S rRNA gene similarity, was Aquamicrobium defluvii from the family 'Phyllobacteriaceae' in the order 'Rhizobiales'. The remaining genera from this order had 16S rRNA gene sequence similarities that were lower than 95.1 % with respect to strain LMG 128. These phylogenetic distances suggested that strain LMG 128 belonged to a different genus. The major fatty acid present in strain LMG 128 was mono-unsaturated straight chain 18 : 1omega7c. The G + C content of the DNA was 53.1 mol%. Strain LMG 128 grew at 4 degrees C but not at 40 degrees C, and tolerated up to 5 % NaCl. The pH range for growth was 6-8. It produced urease and beta-galactosidase, and hydrolysed aesculin. Denitrification was negative. Growth was observed with many carbohydrates as the only carbon source. The data from this polyphasic study indicate that this strain belongs to a new genus of the family 'Phyllobacteriaceae', and therefore it is proposed that strain LMG 128(T) should be reclassified as representing a novel species within the new genus Hoeflea gen. nov., for which the name Hoeflea marina sp. nov. is proposed.