Revealing potential functions of hypothetical proteins induced by genistein in the symbiosis island of Bradyrhizobium japonicum commercial strain SEMIA 5079 (= CPAC 15).

BACKGROUND: Bradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15) is a nitrogen-fixing symbiont of soybean broadly used in commercial inoculants in Brazil. Its genome has about 50% of hypothetical (HP) protein-coding genes, many in the symbiosis island, raising questions about their putative role on the biological nitrogen fixation (BNF) process. This study aimed to infer functional roles to 15 HP genes localized in the symbiosis island of SEMIA 5079, and to analyze their expression in the presence of a nod-gene inducer. RESULTS: A workflow of bioinformatics tools/databases was established and allowed the functional annotation of the HP genes. Most were enzymes, including transferases in the biosynthetic pathways of cobalamin, amino acids and secondary metabolites that may help in saprophytic ability and stress tolerance, and hydrolases, that may be important for competitiveness, plant infection, and stress tolerance. Putative roles for other enzymes and transporters identified are discussed. Some HP proteins were specific to the genus Bradyrhizobium, others to specific host legumes, and the analysis of orthologues helped to predict roles in BNF. CONCLUSIONS: All 15 HP genes were induced by genistein and high induction was confirmed in five of them, suggesting major roles in the BNF process.

Phenotypic switching in Candida tropicalis alters host-pathogen interactions in a Galleria mellonella infection model.

Candida tropicalis is a human pathogen associated with high mortality rates. We have reported a switching system in C. tropicalis consisting of five morphotypes - the parental, switch variant (crepe and rough), and revertant (crepe and rough) strains, which exhibited altered virulence in a Galleria mellonella model. Here, we evaluate whether switching events may alter host-pathogen interactions by comparing the attributes of the innate responses to the various states. All switched strains induced higher melanization in G. mellonella larvae than that induced by the parental strain. The galiomicin expression was higher in the larvae infected with the crepe and rough morphotypes than that in the larvae infected with the parental strain. Hemocytes preferentially phagocytosed crepe variant cells over parental cells in vitro. In contrast, the rough variant cells were less phagocytosed than the parental strain. The hemocyte density was decreased in the larvae infected with the crepe variant compared to that in the larvae infected with the parental strain. Interestingly, larvae infected with the revertant of crepe restored the hemocyte density levels that to those observed for larvae infected with the parental strain. Most of the switched strains were more resistant to hemocyte candidacidal activity than the parental strain. These results indicate that the switch states exhibit similarities as well as important differences during infection in a G. mellonella model.

Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean.

BACKGROUND: The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6T and B. diazoefficiens USDA 110T. RESULTS: Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15. CONCLUSIONS: Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N2 fixation, nodulation and competitiveness.

The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

Reclassification of Rhizobium tropici type A strains as Rhizobium leucaenae sp. nov.

Rhizobium tropici is a well-studied legume symbiont characterized by high genetic stability of the symbiotic plasmid and tolerance to tropical environmental stresses such as high temperature and low soil pH. However, high phenetic and genetic variabilities among R. tropici strains have been largely reported, with two subgroups, designated type A and B, already defined within the species. A polyphasic study comprising multilocus sequence analysis, phenotypic and genotypic characterizations, including DNA-DNA hybridization, strongly supported the reclassification of R. tropici type A strains as a novel species. Type A strains formed a well-differentiated clade that grouped with R. tropici, Rhizobium multihospitium, Rhizobium miluonense, Rhizobium lusitanum and Rhizobium rhizogenes in the phylogenies of the 16S rRNA, recA, gltA, rpoA, glnII and rpoB genes. Several phenotypic traits differentiated type A strains from all related taxa. The novel species, for which the name Rhizobium leucaenae sp. nov. is proposed, is a broad host range rhizobium being able to establish effective root-nodule symbioses with Leucaena leucocephala, Leucaena esculenta, common beans (Phaseolus vulgaris) and Gliricidia sepium. Strain CFN 299(T) ( = USDA 9039(T) = LMG 9517(T) = CECT 4844(T) = JCM 21088(T) = IAM 14230(T) = SEMIA 4083(T) = CENA 183(T) = UMR1026(T) = CNPSo 141(T)) is designated the type strain of Rhizobium leucaenae sp. nov.

Limited vegetative compatibility as a cause of somatic recombination in Trichoderma pseudokoningii

With the aim of a better characterization of the somatic recombination process in Trichoderma pseudokoningii, a progeny from crossings between T. pseudokoningii strains contrasting for auxotroph markers was characterized by RAPD markers and PFGE (electrophoretic karyotype). Cytological studies of the conidia, conidiogenesis and heterokaryotic colonies were also performed. The genotypes of the majority of the recombinant strains analyzed were similar to only one of the parental strains and the low frequency of polymorphic RAPD bands suggested that the nuclear fusions may not occur into the heterokaryon. In some heterokaryotic regions the existence of intensely staining hyphae might be related to cell death. We proposed that a mechanism of somatic recombination other than parasexuality might occur, being related to limited vegetative compatibility after postfusion events, as described for other Trichoderma species.

Limited vegetative compatibility as a cause of somatic recombination in Trichoderma pseudokoningii.

With the aim of a better characterization of the somatic recombination process in Trichoderma pseudokoningii, a progeny from crossings between T. pseudokoningii strains contrasting for auxotroph markers was characterized by RAPD markers and PFGE (electrophoretic karyotype). Cytological studies of the conidia, conidiogenesis and heterokaryotic colonies were also performed. The genotypes of the majority of the recombinant strains analyzed were similar to only one of the parental strains and the low frequency of polymorphic RAPD bands suggested that the nuclear fusions may not occur into the heterokaryon. In some heterokaryotic regions the existence of intensely staining hyphae might be related to cell death. We proposed that a mechanism of somatic recombination other than parasexuality might occur, being related to limited vegetative compatibility after postfusion events, as described for other Trichoderma species.

The nodC, nodG, and glgX genes of Rhizobium tropici strain PRF 81.

Rhizobium tropici is a diazotrophic microsymbiont of common bean (Phaseolus vulgaris L.) that encompasses important but still poorly studied tropical strains, and a recent significant contribution to the knowledge of the species was the publication of a genomic draft of strain PRF 81, which revealed several novel genes [Pinto et al. Funct Int Gen 9:263-270, 2009]. In this study, we investigated the transcription of nodC, nodG, and glgX genes, located in the nod operon of PRF 81 strain, by reverse-transcription quantitative PCR. All three genes showed low levels of transcription when the cells were grown until exponential growth phase in the presence of common-bean-seed exudates or of the root nod-gene inducer naringenin. However, when cells at the exponential phase of growth were incubated with seed exudates, transcription occurred after only 5 min, and nodC, nodG, and glgX were transcribed 121.97-, 14.86-, and 50.29-fold more than the control, respectively, followed by a rapid decrease in gene transcription. Much lower levels of transcription were observed in the presence of naringenin; furthermore, maximum transcription required 8 h of incubation for all three genes. In light of these results, the mechanisms of induction of the nodulation genes by flavonoids are discussed.

Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes.

The genus Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with legumes and non-legumes, and are characterized by physiological and symbiotic versatility and broad geographical distribution. However, despite indications of great genetic variability within the genus, only eight species have been described, mainly because of the highly conserved nature of the 16S rRNA gene. In this study, 169 strains isolated from 43 different legumes were analysed by rep-PCR with the BOX primer, by sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic transcribed spacer (ITS) and by multilocus sequence analysis (MLSA) of four housekeeping genes, glnII, recA, atpD and dnaK. Considering a cut-off at a level of 70 % similarity, 80 rep-PCR profiles were distinguished, which, together with type strains, were clustered at a very low level of similarity (24 %). In both single and concatenated analyses of the 16S rRNA gene and ITS sequences, two large groups were formed, with bootstrap support of 99 % in the concatenated analysis. The first group included the type and/or reference strains of Bradyrhizobium japonicum, B. betae, B. liaoningense, B. canariense and B. yuanmingense and B. japonicum USDA 110, and the second group included strains related to Bradyrhizobium elkanii USDA 76(T), B. pachyrhizi PAC48(T) and B. jicamae PAC68(T). Similar results were obtained with MLSA of glnII, recA, atpD and dnaK. Greatest variability was observed when the atpD gene was amplified, and five strains related to B. elkanii revealed a level of variability never reported before. Another important observation was that a group composed of strains USDA 110, SEMIA 5080 and SEMIA 6059, all isolated from soybean, clustered in all six trees with high bootstrap support and were quite distinct from the clusters that included B. japonicum USDA 6(T). The results confirm that MLSA is a rapid and reliable way of providing information on phylogenetic relationships and of identifying rhizobial strains potentially representative of novel species.

Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean (Phaseolus vulgaris L.) reveals unexpected taxonomic diversity.

The diazotrophic bacteria collectively known as "rhizobia" are important for establishing symbiotic N(2)-fixing associations with many legumes. These microbes have been used for over a century as an environmentally beneficial and cost-effective means of ensuring acceptable yields of agricultural legumes. The most widely used phylogenetic marker for identification and classification of rhizobia has been the 16S rRNA gene; however, this marker fails to discriminate some closely related species. In this study, we established the first multilocus sequence analysis (MLSA) scheme for the identification and classification of rhizobial microsymbionts of common bean (Phaseolus vulgaris L.). We analyzed 12 Brazilian strains representative of a collection of over 850 isolates in addition to type and reference rhizobial strains, by sequencing recA, dnaK, gltA, glnII and rpoA genes. Gene sequence similarities among the five type/reference Rhizobium strains which are symbionts of common bean ranged from 95 to 100% for 16S rRNA, and from 83 to 99% for the other five genes. Rhizobial species described as symbionts of common bean also formed separate groups upon analysis of single and concatenated gene sequences, and clusters formed in each tree were in good mutual agreement. The five additional loci may thus be considered useful markers of the genus Rhizobium; in addition, MLSA also revealed broad genetic diversity among strains classified as Rhizobium tropici, providing evidence of new species.

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains.

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.

Genetic differences between Bradyrhizobium japonicum variant strains contrasting in N(2)-fixation efficiency revealed by representational difference analysis.

Two variant strains of Bradyrhizobium japonicum, derived from SEMIA 566, adapted to the stressful environmental conditions of the Brazilian Cerrados and characterized by contrasting capacities for N(2) fixation, were compared by representational difference analysis (RDA). Twenty-four gene sequences that are unique to the highly effective strain S 370 were identified, eight showing high similarity to known genes, nine encoding putative proteins and seven representing conserved hypothetical or hypothetical proteins; they were classified in eight functional categories. Among those genes, some were highlighted for their known or potential functions in plant-microbe interactions. The nodulation outer protein P (nopP), related to the type-III secretion system (TTSS) and a major determinant of nodulation of some tropical legumes, was detected in the genome of strain S 370. Three coding sequences (CDS) identified by RDA were expressed in proteomics experiments with B. japonicum strain USDA 110 (ChvE and NopP). The use of the sequences identified by RDA in the highly effective strain S 370 might represent an important tool to speed up strain selection programs, accelerating pre-screening procedures. Additionally, the conserved hypothetical and hypothetical proteins identified in strain S 370 might encode important but still unknown proteins related to the symbiosis that deserve further study.

Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah soil.

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N(2)-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.

Variability in Bradyrhizobium japonicum and B. elkanii seven years after introduction of both the exotic microsymbiont and the soybean host in a cerrados soil.

The plasticity of rhizobial genomes is far greater than previously thought, with complex genomic recombination events that may be accelerated by the often stressful environmental conditions of the tropics. This study aimed at evaluating changes in soybean rhizobia due to adaptation to inhospitable environmental conditions (high temperatures, drought, and acid soils) in the Brazilian Cerrados. Both the host plant and combinations of four strains of soybean Bradyrhizobium were introduced in an uncropped soil devoid of rhizobia capable of nodulating soybean. After the third year, seeds were not reinoculated. Two hundred and sixty-three isolates were obtained from nodules of field-grown soybean after the seventh year, and their morphological, physiological, serological, and symbiotic properties determined, followed by genetic analysis of conserved and symbiotic genes. B. japonicum strain CPAC 15 (same serogroup as USDA 123) was characterized as having high saprophytic capacity and competitiveness and by the seventh year represented up to 70% of the cultivable population, in contrast to the poor survival and competitiveness of B. japonicum strain CPAC 7 (same serogroup as CB 1809). In general, adapted strains had increased mucoidy, and up to 43% of the isolates showed no serological reaction. High variability, presumably resulting from the adaptation to the harsh environmental conditions, was verified in rep-PCR (polymerase chain reaction) profiles, being lower in strain CPAC 15, intermediate in B. elkanii, and higher in CPAC 7. Restriction fragment length polymorphism (RFLP)-PCR types of the 16S rDNA corresponded to the following: one type for B. elkanii species, two for B. japonicum, associated to CPAC 15 and CPAC 7, and unknown combinations of profiles. However, when nodC sequences and RFLP-PCR of the nifH region data were considered, only two clusters were observed having full congruence with B. japonicum and B. elkanii species. Combining the results, variability was such that even within a genetically more stable group (such as that of CPAC 15), only 6.4% of the isolates showed high similarity to the inoculant strain, whereas none was similar to CPAC 7. The genetic variability in our study seems to result from a variety and combination of events including strain dispersion, genomic recombination, and horizontal gene transfer. Furthermore, the genetic variability appears to be mainly associated with adaptation, saprophytic capacity, and competitiveness, and not with symbiotic effectiveness, as the similarity of symbiotic genes was higher than that of conserved regions of the DNA.

ABC transporters in Mycoplasma hyopneumoniae and Mycoplasma synoviae: insights into evolution and pathogenicity

ABC transporters represent one of the largest superfamilies of active membrane transport proteins (MTPs) with a highly conserved ATPase domain that binds and hydrolyzes ATP, supplying energy for the uptake of a variety of nutrients and for the extrusion of drugs and metabolic wastes. The complete genomes of a non-pathogenic (J) and pathogenic (7448) strain of Mycoplasma hyopneumoniae, as well as of a pathogenic (53) strain of Mycoplasma synoviae have been recently sequenced. A detailed study revealed a high percentage of CDSs encoding MTPs in M. hyopneumoniae strains J (13.4 percent), 7448 (13.8 percent), and in M. synoviae 53 (11.2 percent), and the ABC systems represented from 85.0 to 88.6 percent of those CDSs. Uptake systems are mainly involved in cell nutrition and some might be associated with virulence. Exporter systems include both drug and multidrug resistant systems (MDR), which may represent mechanisms of resistance to toxic molecules. No relation was found between the phylogeny of the ATPase domains and the lifestyle or pathogenicity of Mycoplasma, but several proteins, potentially useful as targets for the control of infections, were identified.

Genetic characterization of somatic recombination in Trichoderma pseudokoningii

Crossing experiments via hyphal anastomosis between two strains contrasting for auxotrophic markers of Trichoderma pseudokoningii were conducted to characterize the somatic recombination process in this specie. Four crossings were made and a total of 1052 colonies obtained from conidial suspensions of the heterokaryotic colonies were analyzed. Sixty-eight recombinant colonies, from four growing generations, were analyzed for the auxotrophic markers. Of the 68 colonies analyzed, 58 were stable after four generations and the remainders were unstable, reverting to one of the parentals. Most of the recombinant colonies were unstable through subculture and after four growing generations they showed the leu ino met markers (auxotrophic for leucin, inositol and metionin respectively). The unstable recombinant colonies showed irregular growing borders, sparse sporulation and frequent sector formation. The results suggest the occurrence of recombination mechanisms in the heterokaryon (somatic recombination), different from those described for the parasexual cycle or parameiosis. Therefore, we proposed the ocurrence of nuclei degradation from one parental (non prevalent parental) in the heterokaryon and that the resulting chromosomal fragments may be incorporated into whole nuclei of the another parental (prevalent parental). However the parameiosis as originally described cannot be excluded.