End Of 16S rRNA Research Articles

Stenelmis puberula Reitter (Coleoptera: Elmidae)—description of larva and its association with adults, using DNA sequences

The mature larva of Stenelmis puberula Reitter is described for the first time, based on material collected in north-eastern Slovakia (Central Europe). The conspecificity with associated adults was tested using sequences of one nuclear (5' end of 18S rRNA) and three mitochondrial gene fragments (two fragments covering almost the whole cytochrome oxidase c subunit I; and a fragment of cytochrome b) with a total of ca. 2600 bp.

Hedyselmis opis: Description of the larva and its phylogenetic relation to Graphelmis (Coleoptera: Elmidae: Elminae)

The riffle beetle genus Hedyselmis Hinton, 1976 includes two species from the Malay Peninsula, with adults with a highly deviating morphology. Its phylogenetic relationships are unclear, although it has been hypothesized to be related to Graphelmis Deleve, 1968, a large genus widely distributed in the Oriental and East Palaearctic regions. In this paper the larva of H. opis Hinton, 1976 is described based on material collected in the Cameron Highlands (Malaysia) and the conspecificity with co-existing adults tested using sequences of one nuclear (5' end of 18S rRNA) and three mitochondrial gene fragments (5' end of the large ribosomal unit + tRNAleu + 5' end of the NADH dehydrogenase subunit 1; 5' end of cytochrome c oxidase subunit I; and a fragment of cyto- chrome b) with a total of ca. 2,600 bp. This is the first example of the use of molecular data to match different life stages within the family Elmidae. The larva of H. opis has a subcylindrical body typical of many other elmid genera; abdominal segments 1-7 with preserved pleura; and ninth segment with oval operculum. The last instar larvae have clearly visible prominent spiracles on meso- thorax and abdominal segments 1-8. The phylogenetic position of Hedyselmis in relation to Graphelmis was investigated using molecular data for three species of Graphelmis plus a selection of other Elmidae genera. Hedyselmis opis is nested within Graphel- mis, confirming their close relationship and suggesting that their status requires taxonomic revision.

Creation of a Ribosomal Protein Sa/LAMR1 Heterozygous Mouse.

Diamond Blackfan anemia (DBA) is a severe hypoplastic anemia that generally presents early in infancy. Approximately 25% of DBA cases have been linked to mutations in the gene encoding ribosomal protein S19. The remaining cases are of unknown etiology. Our studies in yeast have identified a specific role for Rps19 in the maturation of 40S ribosomal subunits. While only one other small subunit ribosomal protein, Rps18, has a function virtually identical to Rps19 in subunit maturation, several others have functions closely related to Rps19. If the involvement of Rps19 in DBA is linked to its role in the synthesis of 40S ribosomal subunits, we would expect that one or more of these other ribosomal proteins may be responsible for DBA in patients with normal RPS19. To address a potential role for ribosomal proteins other than Rps19 in DBA we have created a transgenic mouse heterozygous at the LAMR1 locus. LAMR1 encodes ribosomal protein Sa, the mammalian homolog of the yeast ribosomal protein S0. Rps0, like Rps19, is required for the maturation of the 3′ end of 18S rRNA. We are currently in the fourth generation of out-crossing the original 129SvEv/C57BL6J chimeras to a C57BL6 background. The frequency of heterozygous pups is about that expected by Mendelian segregation suggesting that haploinsufficiency for ribosomal protein Sa does not lead to significant embryonic lethality. The heterozygous mice do, however, exhibit a higher frequency of craniofacial abnormalities and premature deaths relative to their wild-type littermates. The overall fitness of the heterozygous mice appears to be decreasing with each generation of outcrossing to the C57/BL6 background. Efforts are underway to understand the nature of the premature deaths and to obtain detailed hematological profiles on the LAMR1 heterozygous mice.

Photolabile anticodon stem-loop analogs of tRNAPhe as probes of ribosomal structure and structural fluctuation at the decoding center.

With the recent availability of high-resolution structures of bacterial ribosomes, studies of ribosome-catalyzed protein biosynthesis are now focusing on the nature of conformational changes that occur as the ribosome exerts its complex catalytic function. Photocrosslinking can be relevant for this purpose by providing clues to ribosomal structural fluctuations and dynamics. Here we describe crosslinking experiments on 70S ribosomes using two photolabile anticodon stem-loop derivatives of Escherichia coli tRNAPhe carrying a 4-thiouridine in either position 33 or 37 and denoted Ph-ASLs. One or both of these Ph-ASLs bind to the tRNA A-, P-, and E-sites on the ribosome, with both binding to and photocrosslinking from the E-site showing strong dependence on the presence of a tRNA in the P-site. Both Ph-ASLs crosslink to the extreme 3'-end of 16S rRNA from both the P- and E-sites, providing direct confirmatory evidence in solution for the folding back of the 3'-end toward the decoding region. This suggests that the 3'-end of 16S rRNA may act as a switch in controlling mRNA access to the decoding center, a phenomenon of potential relevance for the translation of leaderless mRNA. E-site bound Ph-ASLs also form photocrosslinks to nucleotides 1395-1398, 1399-1400, and 1491-1494 at the top of helix 44 of 16S rRNA, indicating movement of the decoding center from a position between the A- and P-sites seen in the crystal structure to one neighboring the E-site.

Importance of Transient Structures During Post-transcriptional Refolding of the Pre-23 S rRNA and Ribosomal Large Subunit Assembly

An important step of ribosome assembly is the folding of the rRNA into a functional structure. Despite knowledge of the folded state of rRNA in the ribosomal subunits, there is very little information on the rRNA folding pathway. We are interested in understanding how the functional structure of rRNA is formed and whether the rRNA folding intermediates have a role in ribosome assembly. To this end, transient secondary structures around both ends of pre-23S rRNA were analyzed by a chemical probing approach, using pre-23S rRNA transcripts. Metastable hairpin loop structures were found at both ends of 23S rRNA. The functional importance of the transient structures around the ends of 23S rRNA was tested by mutations that alter only the transient structure. The effect of mutations on 23S rRNA folding was tested in vitro and in vivo. It was found that both stabilization and destabilization of the transient structure around the 5' end of 23S rRNA inhibits post-transcriptional refolding in vitro and ribosome formation in vivo. The data suggest that the transient structure of rRNA has a function during 23S rRNA folding and thereby in ribosome assembly.

The Carboxy-Terminal Extension of Yeast Ribosomal Protein S14 Is Necessary for Maturation of 43S Preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway—the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3′ end of 18S rRNA, consistent with a role for rpS14 in 3′ end processing of 20S pre-rRNA.

Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA.

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3' end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3' end of 18S rRNA and formation of active 40S ribosomal subunits.

The quaternary structure of RNase G from Escherichia coli.

RNase G is the endoribonuclease responsible for forming the mature 5' end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N-terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non-denaturing purifications for overexpressed RNase G. Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit-subunit interface.

Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli.

Era is a small GTP-binding protein and essential for cell growth in Escherichia coli. It consists of two domains: N-terminal GTP-binding and C-terminal RNA-binding KH domains. It has been shown to bind to 16S rRNAs and 30S ribosomal subunits in vitro. Here, we report that a precursor of 16S rRNA accumulates in Era-depleted cells. The accumulation of the precursors is also seen in a cold-sensitive mutant, E200K, in which the mutation site is located in the C-terminal domain. The major precursor molecule accumulated seems to be 17S rRNA, containing extra sequences at both 5' and 3' ends of 16S rRNA. Moreover, the amounts of both 30S and 50S ribosomal subunits relative to the amount of 70S monosomes increase in Era-depleted and E200K mutant cells. The C-terminal KH domain has a high structural similarity to the RbfA protein, a cold shock protein that also specifically associates with 30S ribosomal subunits. RbfA is essential for cell growth at low temperature, and a precursor of 16S rRNA accumulates in an rbfA deletion strain. The 16S rRNA precursor seems to be identical in size to that accumulated in Era mutant cells. Surprisingly, the cold-sensitive cell growth of the rbfA deletion cells was partially suppressed by overproduction of the wild-type Era. The C-terminal domain alone was not able to suppress the cold-sensitive phenotype, whereas Era-dE, which has a 10-residue deletion in a putative effector region of the N-terminal domain, functioned as a more efficient suppressor than the wild-type Era. It was found that Era-dE suppressed defective 16S rRNA maturation, resuming a normal polysome profile to reduce highly accumulated free 30S and 50S subunits in the rbfA deletion cells. These results indicate that Era is involved in 16S rRNA maturation and ribosome assembly.

The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts.

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.

Nob1p is required for cleavage of the 3' end of 18S rRNA.

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.

Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation.

There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving beta-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the beta-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5' untranslated region, it can ensure a substantial enhancing effect on translation.

Genes of de novo pyrimidine biosynthesis from the hyperthermoacidophilic crenarchaeote Sulfolobus acidocaldarius: novel organization in a bipolar operon.

Sequencing a 8,519-bp segment of the Sulfolobus acidocaldarius genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G+C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers, pyrEF-orf8 and pyrBI-orf1-pyrCD-orf2-orf3-orf4, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of pyr genes that also occurs in the recently determined genome sequences of Sulfolobus solfataricus P2 and Sulfolobus tokodaii strain 7; the configuration appears therefore characteristic of Sulfolobus. The quasi-leaderless pyrE and pyrB genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3' end of 16S rRNA. The polycistronic nature of the pyr messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling. pyrB transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate pyrB transcription in bacteria. In contrast, the pyrE-pyrB promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).

Release factor 2 frameshifting sites in different bacteria.

The mRNA encoding Escherichia coli polypeptide chain release factor 2 (RF2) has two partially overlapping reading frames. Synthesis of RF2 involves ribosomes shifting to the +1 reading frame at the end of the first open reading frame (ORF). Frameshifting serves an autoregulatory function. The RF2 gene sequences from the 86 additional bacterial species now available have been analyzed. Thirty percent of them have a single ORF and their expression does not require frameshifting. In the approximately 70% that utilize frameshifting, the sequence cassette responsible for frameshifting is highly conserved. In the E. coli RF2 gene, an internal Shine-Dalgarno (SD) sequence just before the shift site was shown earlier to be important for frameshifting. Mutagenic data presented here show that the spacer region between the SD sequence and the shift site influences frameshifting, and possible mechanisms are discussed. Internal translation initiation occurs at the shift site, but any functional role is obscure.

Structure, expression and products of the ribosomal RNA operons of Rhodopseudomonas palustris No. 7.

Rhodopseudomonas palustris strains carry one or two ribosomal rRNA operons, and those with duplicated rrn operons grow faster. The two rrn operons in R. palustris No. 7 are virtually identical over a 54,70-bp stretch containing the genes for 16S rRNA, tRNAile, tRNAala, 23S rRNA and 5S rRNA, as well as the intergenic spacers and part of the extragenic spacer. In R. palustris, unlike most bacteria with multiple rrn operons, the putative promoter sequences of the two operons are highly diverged, suggesting possible functional differentiation. By simultaneous primer-extension analysis of both pre-rRNAs, we detected a two-fold higher level of expression from rrnA under photoautotrophic conditions. Alteration of the conditions of growth leads to changes in the relative levels of expression of the two operons. Within the 5,470-bp segment, only two sequence differences are found between the 23S rRNA genes; one is at the center of the 23S rRNA molecule and affects a site of unknown function, and the other is within or immediately adjacent to sequences involved in processing of the 5' 23S rRNA IVS. In vitro processing of 5' IVS-containing 23S rRNA precursors from each operon does not reveal any detectable difference between them. The 5' ends of the mature 16S, 23S, and 5S rRNAs were determined by primer-extension analysis, and the 3' end of 23S rRNA was determined by RNA linker ligation-mediated cDNA cloning. The 5' and 3' ends of the R. palustris 23S rRNA molecule are extensively processed, suggesting that, unlike the situation in the established eubacterial model, these ends cannot basepair.

Identification of cis-acting elements involved in 3'-end formation of Saccharomyces cerevisiae 18S rRNA.

In yeast, the 3' end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D. Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site. To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo. Using clustered point mutations, we found that alterations in the sequence spanning site D from position -5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units. More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position -2), whereas sequence changes at position -1, +1, and +2 relative to site D had no effect. The data further demonstrate that the proposed base pairing between the 3' end of 18S rRNA and the 5' end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits. These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation. The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.

6] Escherichia coli ribonuclease G

Publisher Summary The cafA gene, a name used previously for the rng gene encoding Escherichia coli RNase G, was originally found as a putative open reading frame (ORF) of unknown function during analysis of mecillinam-resistant shape determination mutants. Significant sequence similarity is reported between the CafA protein and the N-terminal half of RNase E, which is encoded by the rne gene at 24 min on the E. coli genetic map. This chapter outlines the accumulation of Precursor Molecule of 16S rRNA in cafA :: cat mutant and a two-step maturation of 5′ end of 16S rRNA by RNase E and RNase G. The biological role, if any, of the cytoplasmic axial filament formed by the RNase G protein remains to be determined. When histidine-tagged RNase G proteins were constructed for the purpose of purification, it is observed that some of the constructs did not form cytoplasmic axial filaments by overproduction, but still could complement the defect of 16S rRNA maturation in the rng::cat mutant. This suggests that RNase activity and the ability to form filaments are distinguishable.

Computer analysis of potential stem structures of rRNA operons in various procaryote genomes

The processing of 16S rRNA and 23S rRNA by RNase III in E.coli is known to involve stem structures formed by both ends of the rRNA. Indeed, complementary nucleotide sequences are usually found at both ends of 16S rRNA and 23S rRNA. However, whether or not this phenomenon exists in various other bacteria has not yet been adequately studied. We have conducted computer analyses of potential stem structures of rRNA operons in 12 bacterial and 3 archaeal genomes, and compared characteristics of the stem structures among these species. We systematically computed free energy values by exhaustively ‘annealing’ sequences around the 5′ end and sequences around the 3′ end of both 16S rRNA and 23S rRNA genes, in order to predict potential stem structures.The results suggest that rRNAs in most species form stem structures at both ends. Some species, such as A.aeolicus, seem to form unusually stable stem structures. On the other hand, some rRNAs, such as rRNAs of D.radiodurans, seem not to form solid stem structures. This suggests that rRNA processing in those species must employ a reliable targeting mechanism other than recognizing stem structures by RNase III.

Divergent mechanisms of 5' 23S rRNA IVS processing in the alpha-proteobacteria.

Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group. We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step. Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS. The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R. palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage. Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism. While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs. We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends.

Relationships of bradyrhizobia from Platypodium and Machaerium (Papilionoideae: tribe Dalbergieae) on Barro Colorado Island, Panama.

Enzyme electrophoresis and rRNA sequencing indicated that root nodule bacteria from the canopy tree Platypodium elegans and the lianas Machaerium milleflorum and Machaerium arboreum on Barro Colorado Island, Panama, were highly diverse on a local scale. A total of 11 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 33 isolates analysed. On average, ETs differed from one another at 74% of the 11 enzyme loci assayed, and separate nodules on a single host individual were often occupied by genetically divergent ETs. Certain ETs were sampled multiple times from both Platypodium and Machaerium, suggesting a lack of specificity toward the two legume genera. Within the intervening sequence (IVS) region in the 5' end of 23S rRNA, seven ETs had a length variant similar to that of Bradyrhizobium japonicum USDA 110, and the other four ETs had an IVS region 26-28 bp shorter. Parsimony analysis of both partial 23S rRNA and nearly full-length 16S rRNA sequences indicated that all Platypodium and Machaerium isolates were related to B. japonicum rather than Bradyrhizobium elkanii. The 16S rRNA sequence of one isolate was >99% similar to that of B. japonicum USDA 110, and the closest known relatives for other isolates were Philippine bradyrhizobia from the legumes Stylosanthes and Samanea.