Evolutionary relationships of flavobacterial and enterobacterial endosymbionts with their scale insect hosts (Hemiptera: Coccoidea).

Flavobacteria and Enterobacteriaceae have been previously reported as scale insect endosymbionts. The purpose of this work was twofold: first, to screen different scale insect families for the presence of these endosymbionts by PCR analyses and second, to elucidate the history of cophylogeny between these bacteria and the insects by analysing a portion of 16S rRNA and 18S rRNA gene sequences by two reconciliation tools, CoRe-PA and Jane. From a survey of 27 scale insects within seven families, we identified Flavobacteria and Enterobacteriaceae as coexisting in ten species that belong to the Ortheziidae, Monophlebidae, Diaspididae and Coccidae families, and we frequently found two closely related enterobacteria harboured in the same individual. Analyses performed with CoRe-PA and Jane suggest that Flavobacteria from the scale insects analysed have a unique origin, except for Candidatus Brownia rhizoecola (Flavobacteria of Pseudococcidae, Phenacoccinae), which seems to come from a nonscale insect. Nevertheless, cospeciation between Flavobacteria and scale insects is suggested only within the families Monophlebidae, Ortheziidae and Diaspididae, and host switches seem to have occurred from the ancestors of Monophlebidae and Ortheziidae to insects from families Coccidae, Lecanodiaspididae, Eriococcidae and Pseudococcidae. Our analyses suggest that Enterobacteriaceae underwent more evolutionary events (losses, duplications and host switches), and their phylogenies showed a lower proportion of congruent nodes between host and bacteria, indicating a more relaxed relationship with scale insects compared with Flavobacteria.

Molecular phylogeny of the genus Dactylopius (Hemiptera: Dactylopiidae) and identification of the symbiotic bacteria.

Phylogenetic analyses, from polymerase chain reaction (PCR)-amplified 12S rRNA and 18S rRNA gene sequences from cochineal insects of the genus Dactylopius present in Mexico, showed that D. ceylonicus, D. confusus, and D. opuntiae are closely related. D. coccus constitutes a separate clade, and D. tomentosus is the most distantly related. Bacterial 16S rRNA sequences from all the Dactylopius species sampled showed a common ß-proteobacteria, related to Azoarcus, also found in eggs and in bacteriocytes in D. coccus. We propose the name "Candidatus Dactylopiibacterium carminicum" for this endosymbiont. Other bacterial sequences recovered from the samples were close to those from soil or plant associated bacteria, like Massilia, Herbaspirillum, Acinetobacter, Mesorhizobium, and Sphingomonas, suggesting a possible horizontal transmission from Cactaceae plant sap to Dactylopius spp. during feeding. This is the first molecular analysis of Dactylopius species and of their associated bacteria.

Natural endophytic association between Rhizobium etli and maize (Zea mays L.).

Maize (Zea mays) and bean (Phaseolus vulgaris) have been traditionally grown in association for thousands of years in Mesoamerica. From surface sterilized maize roots, we have isolated over 60 Rhizobium strains that correspond to Rhizobium etli bv. phaseoli (the main symbiont of bean) on the basis of 16S rRNA gene restriction patterns, metabolic enzyme electropherotypes, organization of nif genes, and the ability to nodulate beans. The colonization capacity of some of the isolates was tested with an unimproved maize cultivar and with 30 maize land races. Increases in plant dry weight upon R. etli inoculation were recorded with some of the land races, and these increases may be related to plant growth promotion effects. Additionally, from within maize grown in monoculture we have also recovered R. etli isolates recognizable by their 16S rRNA gene types, which lack nif genes and are incapable of nodulating bean. These strains are presumed to correspond to the earlier described non-symbiotic R. etli obtained from bean rhizosphere.

Novel nitrogen-fixing acetic acid bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter azotocaptans sp. nov., associated with coffee plants.

Diazotrophic bacteria were isolated, in two different years, from the rhizosphere and rhizoplane of coffee (Coffea arabica L.) plants cultivated in Mexico; they were designated as type DOR and type SAd isolates. They showed characteristics of the family Acetobacteraceae, having some features in common with Gluconacetobacter (formerly Acetobacter) diazotrophicus, the only known N2-fixing species of the acetic acid bacteria, but they differed from this species with regard to several characteristics. Type DOR isolates can be differentiated phenotypically from type SAd isolates. Type DOR isolates and type SAd isolates can both be differentiated from Gluconacetobacter diazotrophicus by their growth features on culture media, their use of amino acids as nitrogen sources and their carbon-source usage. These results, together with the electrophoretic mobility patterns of metabolic enzymes and amplified rDNA restriction analysis, suggested that the type DOR and type SAd isolates represent two novel N2-fixing species. Comparative analysis of the 16S rRNA sequences revealed that strains CFN-Cf55T (type DOR isolate) and CFN-Ca54T (type SAd isolate) were closer to Gluconacetobacter diazotrophicus (both strains had sequence similarities of 98.3%) than to Gluconacetobacter liquefaciens, Gluconacetobacter sacchari (similarities < 98%) or any other acetobacteria. Strain CFN-Cf55T exhibited low levels of DNA-DNA reassociation with type SAd isolates (mean 42%) and strain CFN-Ca54T exhibited mean DNA-DNA reassociation of 39.5% with type DOR isolates. Strains CFN-Cf55T and CFN-Ca54T exhibited very low DNA reassociation levels, 7-21%, with other closely related acetobacterial species. On the basis of these results, two novel N2-fixing species are proposed for the family Acetobacteraceae, Gluconacetobacter johannae sp. nov. (for the type DOR isolates), with strain CFN-Cf55T (= ATCC 700987T = DSM 13595T) as the type strain, and Gluconacetobacter azotocaptans sp. nov. (for the type SAd isolates), with strain CFN-Ca54T (= ATCC 70098ST = DSM 13594T) as the type strain.

Identification of a system that allows a Rhizobium tropici dctA mutant to grow on succinate, but not on other C4-dicarboxylates.

A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899. This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source. Our results suggest the presence of a novel succinate-specific transport system in R. tropici. Biochemical characterization indicated that this alternative transport system in GAI is active and dependent on an energized membrane. It was also induced by succinate and aspartate, and was repressed by glucose and glycerol. Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation. Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.

Limited genetic diversity of Brucella spp.

Multilocus enzyme electrophoresis (MLEE) of 99 Brucella isolates, including the type strains from all recognized species, revealed a very limited genetic diversity and supports the proposal of a monospecific genus. In MLEE-derived dendrograms, Brucella abortus and a marine Brucella sp. grouped into a single electrophoretic type related to Brucella neotomae and Brucella ovis. Brucella suis and Brucella canis formed another cluster linked to Brucella melitensis and related to Rhizobium tropici. The Brucella strains tested that were representatives of the six electrophoretic types had the same rRNA gene restriction fragment length polymorphism patterns and identical ribotypes. All 99 isolates had similar chromosome profiles as revealed by the Eckhardt procedure.

Sesbania herbacea-Rhizobium huautlense Nodulation in Flooded Soils and Comparative Characterization of S. herbacea-Nodulating Rhizobia in Different Environments.

The nodulation of S. herbacea was compared under flooded and non-flooded conditions in two different soils. One soil was from a flooded field in Sierra de Huautla, the native habitat of this legume, while the other soil was from a well-drained field in Cuernavaca, where rhizobia were found to nodulate the introduced S. herbacea plants. Nodulation of the plants was completely eliminated by flooding in the Cuernavaca soil, whereas nodules were obtained in the same soil under non-flooded conditions. In contrast, nodules were formed in Huautla soil under both flooded and non-flooded conditions. Most isolates, except isolate HS2, from Huautla soil and water were identified as R. huautlense by colony morphology, growth rate, PCR-RFLP of 16S rRNA genes, MLEE, cellular plasmid contents, and RFLP of nifH and nodDAB genes. Isolate HS2 was identified as Mesorhizobium sp. Isolates from Cuernavaca soil were different from R. huautlense in many aspects and were classified into five rDNA types within the genera Mesorhizobium, Rhizobium, and Sinorhizobium by PCR-RFLP of 16S rRNA genes. R. huautlense is a water Rhizobium species. Growth by denitrification under oxygen limitation or with ethanol was observed for R. huautlense bacteria but not for the isolates from Cuernavaca. In an interstrain nodulation competitive assay under both flooded and non-flooded conditions, R. huautlense strain S02 completely inhibited the nodulation of Mesorhizobium sp. Sn2, an isolate from Cuernavaca. From these results, we conclude that R. huautlense has the unique ability to nodulate S. herbacea not only in flooded soils, but in non-flooded soils as well.

Rhizobium etli bv. mimosae, a novel biovar isolated from Mimosa affinis.

Fifty rhizobial isolates from root nodules of Mimosa affinis, a small leguminous plant native to Mexico, were identified as Rhizobium etli on the basis of the results of PCR-RFLP and RFLP analyses of small-subunit rRNA genes, multilocus enzyme electrophoresis and DNA-DNA homology. They are, however, a restricted group of lineages with low genetic diversity within the species. The isolates from M. affinis differed-from the R. etli strains that orginated from bean plants (Phaseolus vulgaris) in the size and replicator region of the symbiotic plasmid and in symbiotic-plasmid-borne traits such as nifH gene sequence and organization, melanin production and host specificity. A new biovar, bv. mimosae, is proposed within R. etli to encompass Rhizobium isolates obtained from M. affinis. The strains from common bean plants have been designated previously as R. etli bv. phaseoli. Strains of both R. etli biovars could nodulate P. vulgaris, but only those of bv. mimosae could form nitrogen-fixing nodules on Leucaena leucocephala.

Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and description of Mesorhizobium amorphae sp. nov.

Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-fixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplified 16S rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 16S rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16S rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 16S rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 16S rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov.

Three replicons of Rhizobium sp. Strain NGR234 harbor symbiotic gene sequences.

Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b.

Rhizobium huautlense sp. nov., a symbiont of Sesbania herbacea that has a close phylogenetic relationship with Rhizobium galegae.

The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve at the Sierra de Huautla, Mexico, were isolated and characterized. All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 16S rRNA genes as revealed by PCR-RFLP analysis. Similarity in the sequences of the 16S rRNA genes placed the isolates on a phylogenetic branch shared with R. galegae. Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R. galegae. A new species Rhizobium huautlense, represented by the Sesbania isolate SO2T, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 16S rRNA genes, and their distinct ETs compared with R. galegae. The description of R. huautlense is significant because in the reconstruction of the phylogeny at R. huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R. galegae. The revised phylogenetic tree would tend to indicate common ancestry between R. galegae and Rhizobium leguminosarum.

Generation of Rhizobium strains with improved symbiotic properties by random DNA amplification (RDA)

To select for bacterial strains with enhanced phenotypes, random fragments of a whole genome, or a defined region of the genome, are cloned in a nonreplicating vector. The resulting plasmids are integrated by recombination into the homologous DNA region of the original strain. Integration gives rise to a nontandem direct duplication of the corresponding DNA region separated by the vector moiety of the plasmid. Recombination between the direct repeats leads to tandem duplication and further amplification of the entire integrated DNA, including the vector. Bacteria harboring the amplified DNA are selected by increasing the dosage of an antibiotic corresponding to a resistance marker of the integrated vector. Pooled strains carrying amplifications are then challenged with a selective pressure for the desired phenotype. After repeated selection cycles, the most fit strains are isolated. We used this process, which we called random DNA amplification, to select Rhizobium strains with increased competitiveness for nodule formation. Derivatives containing randomly amplified DNA regions of the symbiotic plasmid of Rhizobium tropici CFN299 strain were generated. Pools of amplified strains were inoculated onto various tropical legumes. After several cycles of selection through plants, amplified derivatives showing an increased competitiveness for nodule formation with the leguminous plant Macroptilium atropurpureum were obtained.

Limited Genetic Diversity in the Endophytic Sugarcane Bacterium Acetobacter diazotrophicus.

Acetobacter diazotrophicus isolates that originated from different sugarcane cultivars growing in diverse geographic regions of Mexico and Brazil were shown to have limited genetic diversity. Measurements of polymorphism in the electrophoretic mobilities of metabolic enzymes revealed that the mean genetic diversity per enzyme locus (among the four electrophoretic types distinguished) was 0.064. The results of the genetic analysis indicate that the genetic structure of A. diazotrophicus is clonal, with one largely predominant clone. Plasmids were present in 20 of 24 isolates, and the molecular sizes of the plasmids ranged from 2.0 to 170 kb. Two plasmids (a 20- to 24-kb plasmid detected in all 20 plasmid-containing isolates and a 170-kb plasmid observed in 14 isolates) were highly conserved among the isolates examined. Regardless of the presence of plasmids, all of the isolates shared a common pattern of nif structural gene organization on the chromosome.