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Numerical analysis of phenotypic properties, genomic fingerprinting, and multilocus sequence analysis of Bradyrhizobium strains isolated from root nodules of Lembotropis nigricans of the tribe Genisteae

Numerical analysis of phenotypic properties, genomic fingerprinting, and multilocus sequence... Purpose The aim of this study was to estimate the level of genomic and phenotypic diversity as well as the genus and species position of bacterial strains isolated from root nodules of Lembotropis nigricans (family Fabaceae). Methods The genomic diversity of studied L. nigricans nodule symbionts was examined by using BOX-PCR and AFLP (amplified fragment length polymorphism) fingerprinting techniques. To assign bacteria to the genus, numerical analysis of phenotypic features and comparative analysis of 16S rDNA sequences were performed. The comparative analysis of combined atpD, dnaK, gyrB,and rpoB gene sequences (multilocus sequence analysis, MLSA) was used to determine the most closely related species to the studied bacteria. Results Both BOX-PCR and AFLP techniques revealed a high level of genomic heterogeneity of L. nigricans nodulators. Among 33 studied bacteria, 32 genotypes were delineated by the AFLP method and 27 genotypes were identified by the BOX-PCR fingerprinting. The numerical analysis of 86 phenotypic characteristics of L. nigricans nodule isolates and reference rhizobia showed that studied bacteria belong to the genus Bradyrhizobium.Affiliation of L. nigricans nodule isolates to the genus Bradyrhizobium was supported by comparative analysis of 16S rDNA sequences and the concatenation of atpD, dnaK, gyrB,and rpoB gene sequences. MLSA indicated also that L. nigricans microsymbionts are members of Bradyrhizobium japonicum. Conclusion L. nigricans root nodule symbionts are members of Bradyrhizobium japonicum and exhibit high phenotypic and genomic diversity important for their survival in soil. . . . . . Keywords AFLP BOX-PCR Bradyrhizobium Genisteae Lembotropis nigricans Multilocus sequence analysis Introduction Rhizobia are soil-inhabiting bacteria capable of establishing nitrogen-fixing symbiosis with plant species of the family Fabaceae. Genisteae encompassing more than 600 species Magdalena Wójcik and Michał Kalita contributed equally to this work. distributed among 25 genera is one of the largest tribes within The GenBank accession numbers for sequences generated in this study thelegumefamily (Cardoso et al. 2013). There are published are as follows: MK183761-MK183771 (16S rRNA), MK202810- reports on the phylogeny and taxonomy of bacterial strains MK202820 (atpD), MK202821-MK202831 (dnaK), MK202832- isolated from root nodules of 77 plant species of the tribe MK202842 (gyrB), MK202843-MK202853 (rpoB). Genisteae. Based on these reports, it was concluded that the Electronic supplementary material The online version of this article strains of the genus Bradyrhizobium form a predominant (https://doi.org/10.1007/s13213-019-01491-6) contains supplementary group infecting Genisteae legumes (Stępkowski et al. 2018). material, which is available to authorized users. Still, there are no data available on rhizobia of 14 Genisteae genera. One of such plant genus is Lembotropis comprising * Michał Kalita michal.kalita@umcs.pl two species: Lembotropis nigricans (L. Griseb.) and Lembotropis emeriflora (Rchb.). L. nigricans (black broom) is distributed in Central and Southeastern Europe; however, its Department of Genetics and Microbiology, Maria Curie Sklodowska University, Akademicka 19, 20-033 Lublin, Poland exact range is hard to estimate since it has been grown as an 1124 Ann Microbiol (2019) 69:1123–1134 ornamental shrub for many years and it can currently be found were used for root nodule collection. The rhizobia studied in many areas as a wild plant (Petrowicz 1981). Although it were isolated from root nodules with the standard procedure has been previously demonstrated that L. nigricans produces (Kalita and Małek 2004). Yeast extract mannitol medium cylindrical root nodules (Łotocka et al. 2012), the bacteria (YEM) was routinely used for cultivation of the rhizobia at residing in these structures have not been analyzed for their 28 °C. The strains were stored as source cultures on YEM phenotypic properties, genomic diversity, and taxonomic slants at 4 °C. affiliation. Polyphasic taxonomy, which integrates all available geno- Phenotypic characterization and numerical analysis typic, phenotypic, and phylogenetic information, has been of phenotypic properties widely used for identification and classification of bacteria since the 1970s (Vandamme and Peeters 2014). Numerical L. nigricans nodule isolates were examined for cell morphol- analysis of phenotypic and genotypic features allows differen- ogy, motility, and generation time according to a procedure tiation of closely related bacteria and preliminary determina- described earlier by Wdowiak and Małek (2000). Their phe- tion of the genus position of microorganisms. The PCR fin- notypic properties, i.e., temperature and pH growth ranges, gerprinting techniques such as BOX-PCR and AFLP are often NaCl tolerance, acid and alkali production in YEM agar sup- used for the assessment of the genomic diversity of rhizobia plemented with bromothymol blue as a pH indicator, utiliza- isolated from root nodules of different legume species (Kalita tion of different compounds as sole carbon and nitrogen and Małek 2006; Liu et al. 2011; Gnatetal. 2015;Xu et al. sources, resistance to antibiotics and dyes, reaction in litmus 2016; Wdowiak-Wróbel et al. 2017; Chidebe et al. 2018). milk, activity of urease, catalase, β-galactosidase, phospha- Since the 1990s, the phylogenetic studies of root nodule tase, nitrate reductase, cytochrome oxidase, β-D-glucosidase, isolates have been based mainly on partial or complete 16S and peroxidase, synthesis of indole, melanin, and indole-3- rDNA sequences (Young et al. 1991; Vinuesa et al. 1998). acetic acid (IAA), adsorption of Congo red and Calcofluor, Since then, many studies have demonstrated that the diversity and precipitation of calcium glycerophosphate were deter- in the 16S rRNA sequences of rhizobia is low and distin- mined according to Wdowiak and Małek (2000). The utiliza- guishes species poorly (Menna et al. 2009;Delamutaet al. tion of various compounds as a sole carbon source was studied 2013). Due to the limitations of the 16S rRNA gene analysis, in modified Bergersen’s synthetic medium (BS medium) in protein-encoding genes with evolution rates faster than that of which mannitol was replaced by the tested compound. The 16S rDNA, but conserved sufficiently to retain genetic infor- utilization of various compounds as a sole nitrogen source mation, have been proposed as alternative phylogenetic was examined in the same BS medium with the tested sub- markers. atpD, dnaK, glnII, gyrB, recA,and rpoB are exam- stance instead of NH Cl. All phenotypic tests were done in ples of housekeeping genes that are frequently used in tracing triplicate and repeated two times. The results were scored after the evolutionary history of bacteria (Gaunt et al. 2001; 5 days for fast-growing strains and 7–10 days for slow- Stępkowski et al. 2003;Vinuesa etal. 2005; Rivas et al. growing strains (Garrity et al. 2005). 2009; Kalita and Małek 2017; Huang et al. 2018). For numerical analysis, the phenotypic features of the In this study, we examined the genus position and evolu- rhizobia studied were coded in the binary system. Next, the tionary history of L. nigricans root nodule isolates using nu- simple matching similarity coefficient (SM) of each strain pair merical analysis of phenotypic properties and comparative was estimated and a similarity matrix was generated (Sneath sequence analysis of 16S rRNA, atpD, dnaK, gyrB,and and Sokal 1973). Based on the similarity matrix, clustering rpoB genes. L. nigricans symbionts were also examined for analysis was performed with the unweighted pair group meth- BOX-PCR and AFLP patterns to study their genomic relation- od with arithmetic mean (UPGMA) using the NTSYSpc soft- ship and diversity. ware package (Exeter Software). Isolation of total genomic DNA Materials and methods For DNA isolation, bacteria were grown in 30 ml of liquid Bacterial strains YEM medium for 4–5 days at 28 °C. DNA was extracted and purified according to the method proposed by Pitcher et al. The strains used in this study and their origin are listed in (1989) as described elsewhere (Kalita and Małek 2004). Supplementary Table S1.Rootnodules of L. nigricans were collected from plants growing at a single location in southeast BOX-PCR, AFLP, and data analysis Poland (51° 08′ 47.2″ N23° 24′ 43.3″ E). The L. nigricans population occupied area of approximately 50 m . Ten ran- The primers and PCR cycling conditions used in the BOX- domly selected individual plants from five distinct patches PCR and AFLP analyses are listed in Supplementary 1125 Ann Microbiol (2019) 69:1123–1134 Table S2. The AFLP procedure was done as described by Results and discussion Tyrka (2002) with some small modifications as described else- where (Kalita and Małek 2006). Each reaction was repeated Phenotypic properties of L. nigricans rhizobia three times on the same DNA matrix. Only informative and reproducible products of the genomic fingerprinting reactions A total of 48 bacterial strains, including 33 L. nigricans were analyzed. The PCR products were separated in 1.5% symbionts and 15 reference strains, representing different agarose gel, stained with ethidium bromide, and visualized species of the genera: Bradyrhizobium, Ensifer, under UV light. The DNA profiles yielded by the BOX- Mesorhizobium,and Rhizobium were analyzed for 86 phe- PCR and AFLP methods were analyzed using BIO-GEN pro- notypic properties. Table 1 presents some physiological gram version 11.01 (Vilber-Lourmat). The strains were and metabolic properties of the tested bacteria. In grouped by the Nei and Li coefficient (Nei and Li 1979)and Supplementary Table S3, the results of all 86 characteris- the dendrogram was constructed using the UPGMA method. tics are shown. The growth temperature range for the black broom rhizobia was determined to be between 13 and 3 °C Amplification and sequencing of 16S rRNA, atpD, with an optimal temperature of 28–30 °C, typical for most dnaK, gyrB,and rpoB genes rhizobia. All L. nigricans symbionts, likewise the reference Bradyrhizobium species, grew at pH 5–8. No strain toler- The primers and PCR amplification conditions used in this ated pH 4. Forty-five percent of the strains were able to study are listed in Supplementary Table S2. All PCR amplifi- grow at pH 9 and 21% grew even at pH 10. The black cation reactions were carried out with ReadyMix™ Taq PCR broom rhizobia were able to grow on YEM medium with Reaction Mix (Sigma) according to the manufacturer’srecom- 0.5 and 1% NaCl. Most of them tolerated 2% sodium chlo- mendations. DNA-free water was used in negative PCR con- ride and 32% of the studied isolates tolerated 3% NaCl, in trols. Genomic DNA of B. japonicum USDA6 wasusedasa contrast to the genus Bradyrhizobium strains, which gen- template in positive PCR controls. The amplified products erally exhibit high sensitivity to salinity (Garrity et al. were purified with Clean-Up or Gel-Out purification columns 2005). The L. nigricans symbionts were slow-growing (A&A Biotechnology) and sequenced with the BigDye rhizobia with generationtime~6hinYEM brothat Terminator Cycle sequencing kit (Thermo Fisher Scientific) 28 °C. Such a doubling time is characteristic for the strains using the 3500 Genetic Analyzer according to the manufac- of the genus Bradyrhizobium (van Berkum and Eardly turer’s procedures. 1998). Tests for assimilation of different compounds as sole carbon and nitrogen sources are commonly used in Phylogenetic analysis taxonomic studies of rhizobia. In our experiments, 25 dif- ferent carbon-containing compounds were tested as sole The sequences yielded in this study were compared to the sources of carbon for bacteria. All the tested isolates uti- nucleotide sequences from GenBank database using the lized 12 of the 25 studied carbon sources, i.e., L-alanine, L- BLASTprogram (Altschuletal. 1990). Since all the se- arginine, dextrin, mannitol, glycerol, sucrose, Tween-20, quences were most similar to the sequences of the genus D-fructose, D-galactose, insulin, D-xylose, and sodium tar- Bradyrhizobium strains, only reference bradyrhizobia were trate. The other carbon compounds served as growth sub- included for further phylogenetic analyses. All phylogenetic strates for only some bacteria studied (Table 1). In YEM analyses were conducted in MEGA 7 (Kumar et al. 2016)as medium with mannitol as a sole carbon source, black follows. Multiple sequence alignments were constructed and broom symbionts synthesized acid products similar to the resulting alignments were corrected manually. To obtain Rhizobium, Ensifer,and Mesorhizobium species strains the same number of analyzed positions in the alignment, lon- (Garrity et al. 2005;Gnatetal. 2014). Most of the tested ger sequences were truncated. As a result, 1228, 429, 204, strains did not utilize disaccharides such as maltose and 563, and 452 nucleotide positions were analyzed for the 16S trehalose similar to slow-growing bradyrhizobia, which rRNA, atpD, dnaK, gyrB,and rpoB genes, respectively. The generally do not have disaccharide uptake systems maximum likelihood (ML) method was used to reconstruct (Glenn and Dilworth 1981;Elkan 1992). However, most the phylogeny of the analyzed genes. jModelTest (Darriba of them exhibited good growth on lactose and sucrose. et al. 2012) was used to choose the best-fit evolutionary model Of the 21 nitrogen sources tested, only DL-ornithine was for each studied gene. To determine the degree of the statisti- not utilized by all the L. nigricans symbionts, whereas 81 cal support for the branches in the phylogeny, 1000 bootstrap and 79% of the bacteria did not utilize L-lysine and sodium replicates of the data were analyzed. Sequence identity values hippurate, respectively. The relevant phenotypic traits that for atpD-dnaK-gyrB-rpoB concatenation were calculated differentiated the L. nigricans nodulators and the reference using BioEdit software (Hall 2011) based on the multiple Bradyrhizobium species are listed in Table 1. An important alignment constructed in MEGA7. taxonomic criterion used in differentiation of fast-growing 1126 Ann Microbiol (2019) 69:1123–1134 Table 1 Some phenotypic characteristics of Lembotropis nigricans root nodule isolates and reference Bradyrhizobium strains Characteristics Lembotropis Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium nigricans elkanii USDA liaoningense USDA yuanmingense CCBAU diazoefficiens USDA japonicum USDA a T T T T T isolates (n =33) 76 (n =1) 3622 (n =1) 10071 (n =1) 110 (n =1) 6 (n =1) Carbon sources used b c D-Arabinose + (24) ++ + + + D-Cellobiose + (21) + + + + + D-Glucose + (27) + + + + + D-Raffinose + (9) + + + + + D-Trehalose + (4) – ++ –– D-Xylose + (33) + –– ++ Dextrin + (33) –– ++ + Inulin + (33) + –– ++ Lactose + (27) –– – ++ L-Alanine + (33) + + – ++ L-Arginine + (33) + –– ++ L-Asparagine + (24) + –– ++ L-Glutamine + (29) + + + + + L-Lysine + (14) –– – – – L-Rhamnose + (14) + + + + + L-Tyrosine + (29) + –– – – Maltose + (9) – + – ++ Salicin + (21) + – ++ + Sodium citrate + (24) + –– ++ Sodium +(4) –– – – – hippurate Starch + (24) –– – – – Nitrogen sources used DL-Isoleucine + (30) + – + – + L-Glutamic acid + (33) + + – ++ L-Lysine + (6) + – + – + L-Phenylalanine + (29) + –– ++ L-Serine + (30) + –– ++ Sodium + (7) + –– – – hippurate Sodium nitrate + (31) + + + + + Resistant to pH 9.0 + (15) –– – – – pH 10.0 + (7) –– – – – 1.0% NaCl + (32) + –– – – 3.0% NaCl + (11) –– – – – Auramine +(23) + – ++ + −1 0.05 μgml Crystal violet +(2) –– – ++ −1 0.1 μgml Methyl green +(5) –– – – – −1 0.13 μgml Neutral red + (24) – ++ + + −1 0.2 μgml Ampicillin +(22) + –– ++ −1 100 μgml Ampicillin +(13) + –– ++ −1 200 μgml Rifampicin +(28) + + + + + −1 20 μgml Ann Microbiol (2019) 69:1123–1134 1127 Table 1 (continued) Characteristics Lembotropis Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium nigricans elkanii USDA liaoningense USDA yuanmingense CCBAU diazoefficiens USDA japonicum USDA a T T T T T isolates (n =33) 76 (n =1) 3622 (n =1) 10071 (n =1) 110 (n =1) 6 (n =1) Rifampicin + (12) –– – ++ −1 200 μgml Streptomycin +(31) + + + + + −1 10 μgml Streptomycin +(13) + –– – – −1 200 μgml Tetracycline +(30) + + + + + −1 10 μgml Tetracycline + (3) + –– – – −1 40 μgml n, the number of studied strains +, −, strains were positive, negative, respectively Value in parentheses is the number of strains with positive reaction from slow-growing rhizobia is their tolerance to antibiotics BOX-PCR and AFLP analysis (Elkan 1992). The black broom nodule isolates showed rather high tolerance to the tested antibiotics, similar to In recent years, many methods have been developed for mo- the Bradyrhizobium species (Kalita and Małek 2004). lecular typing and assessing genomic heterogeneity of bacte- −1 Most of them were resistant to ampicillin (100 μgml ), ria mainly within species or within closely related species. −1 −1 rifampicin (20 μgml ), streptomycin (10 μgml ), and One of the best known genome fingerprinting techniques −1 tetracycline (10 μgml ). All of them were able to grow in based on polymerase chain reaction (PCR) is BOX-PCR of the medium supplemented with acridine orange (0.013%), highly conserved repetitive DNA sequences occurring natu- crystal violet (0.013%), methyl red (0.05%), methyl green rally in bacterial genomes (Lupski and Weinstock 1992; (0.065%), neutral red (0.1%), nigrosine (0.5%), and safra- Versalovic et al. 1994) as well as AFLP based on nucleotide nin (0.2%). Rhizobia specific to L. nigricans were highly changes within restriction sites and adjacent bases, which homogeneous in enzyme activities (except catalase and serve for primer annealing in the PCR reaction (Blears et al. phosphatase) and all of them hydrolyzed urea, reduced ni- 1998; Savelkoul et al. 1999). Both techniques generating trate, produced active cytochrome oxidase, and exhibited DNA profiles specific to a given genome were used for anal- evident alkaline reaction in the litmus milk test. ysis of the genomic diversity and genomic relationship of the The phenotypic properties of the L. nigricans rhizobia and 33 L. nigricans root nodule isolates. In the BOXA1R-PCR the reference strains representing the genera method, a 22-base oligonucleotide primer containing 68% of Bradyrhizobium, Ensifer, Mesorhizobium,and Rhizobium GC was applied. The use of the GC-rich primer in the BOX- were subjected to numerical analysis with the use of the PCR method was associated with the high GC content of NTSYSpc software. The resulting dendrogram is shown in rhizobial genomes (Garrity et al. 2005) and helped to maxi- Fig. 1. The complete binary matrix table used for the numer- mize the number of amplicons. In the PCR reaction with all ical analysis is available as Supplementary Table 3. On the black broom rhizobia, the BOXA1R primer produced DNA basis of the cluster analysis, all strains included into analysis bands ranging in the size from 275 to 2134 nucleotides, with formed two phena at the similarity coefficient level of 0.71. an average number of nine bands per strain. The DNA BOX Phenon I included bacteria of the genera Rhizobium, Ensifer, patterns were used for cluster analysis and presentation of the and Mesorhizobium. Phenon II contained slow-growing genomic relationship in the form of a dendrogram (Fig. 2). In strains of the genus Bradyrhizobium and L. nigricans sym- the generated dendrogram, the 33 L. nigricans symbionts were bionts, which formed a separate subgroup at a similarity divided into three main clusters. In the first cluster, two strains coefficient of 0.82. The results presented in the dendrogram (LN1 and LN14) were placed distinctly from the other bacte- indicate that the slow-growing black broom rhizobia belong ria. The second main cluster comprised 17 isolates with a to the genus Bradyrhizobium. The high level of phenotypic DNA pattern similarity level from 71 to 100%, whereas the diversity of the L. nigricans symbionts determined by the third one encompassed 14 strains with a DNA pattern similar- numerical analysis may facilitate their survival in changing ity level in the range from 82 to 100%. The BOX-PCR method environmental conditions in which these bacteria naturally allowed identification of 27 genomotypes among the 33 black live. broom microsymbionts. Eleven strains belong to 5 clusters 1128 Ann Microbiol (2019) 69:1123–1134 Fig. 1 UPGMA dendrogram showing phenotypic relationships among 33 Lembotropis nigricans root nodule isolates and reference strains comprising at least two root nodule isolates sharing identical AFLP profiles of the bacteria studied contained from 1 to 19 DNA profiles (Fig. 2). DNA bands per strain and their size was from 184 to 1486 bp. To investigate the genomic diversity of the L. nigricans On the basis of DNA banding profiles, it was possible to symbionts in greater detail, the AFLP technique was also differentiate all black broom rhizobia except two strains, used. Presently, bacteriologists frequently use this method in which exhibited identical genomic patterns in the AFLP meth- the assessment of genomic diversity both within species and od with the PsI-GC primer. The genomic relationships of across different bacterial populations (Aserse et al. 2012;Li L. nigricans symbionts based on AFLP fingerprinting data et al. 2012;Xuet al. 2016). For fingerprinting the black broom are presented in the dendrogram generated by UPGMA cluster rhizobial genomes and assessment of their genomic relation- analysis (Fig. 2). In this tree, the rhizobia studied were split ships with the AFLP method, PstI endonuclease recognizing a into two major groups at a DNA similarity coefficient level of GC-rich sequence 5′-CTGGAG-3′ and the primer pair PstI- 0.55. One cluster encompassed ten rhizobia, including two GC with GC as arbitrary bases at the 3′ end were used. The strains (LN22 and LN23) sharing identical DNA patterns. 1129 Ann Microbiol (2019) 69:1123–1134 Fig. 2 Dendrograms based on (a) BOX-PCR and (b)AFLP dataof 33 Lembotropis nigricans root nodule isolates. Nei and Li coef- ficient was used for similarity measure. UPGMA was used for clustering The other group comprised 23 strains separated into two Guerrouj et al. 2013; Grönemeyer et al. 2017; Costa et al. genomically different subgroups at a similarity coefficient lev- 2018). el of 0.62. Genome heterogeneity of rhizobia is shaped by In order to clarify the genus position of the L. nigricans environmental factors and limited by the symbiotic interaction symbionts and investigate their evolutionary relationship with with the host plant. Our studies showed that the AFLP tech- other bacteria of the genus Bradyrhizobium, the nearly full- nique was superior to the BOX-PCR method in differentiating length 16S rRNA encoding genes of 11 symbionts the L. nigricans symbionts and facilitated identification of 32 representing different phenotypic and genomic groups of genomotypes among the 33 bacterial strains studied. We also black broom nodule isolates were amplified and sequenced. showed that both these genome profiling techniques used of- The 16S rDNA sequences of the studied rhizobia were aligned fer a convenient way to choose the representative strains from and compared with those of other nodule bacteria available in each genomic group for further taxonomic studies, such as the GenBank database. The evolutionary distances between 16S rDNA and multilocus sequence analysis (MLSA). L. nigricans symbionts and reference bacteria representing the genus Bradyrhizobium were calculated from a 1228 bp- long alignment and molecular phylogeny was reconstructed 16S rDNA sequence analysis using the maximum likelihood method (ML). The level of sequence similarity between the 16S rDNA of the black Comparative analysis of 16S rDNA sequences is widely used broom rhizobia and those of Bradyrhizobium ranged from to study the taxonomic position of bacteria at the genus level 94.8 to 100%. The L. nigricans rhizobia were most similar and to depict bacterial phylogeny. It was demonstrated that a (≥ 99.5%) in their 16S rDNA sequences to B. japonicum T T 16S rRNA gene sequence similarity lower than 98.7% sug- USDA 6 , B. canariense BTA-1 , B. liaoningense USDA T T gests that bacterial strains belong to distinct species (Yarza 3622 , B. daqingense CCBAU 15774 ,and B. americanum et al. 2014). Analysis of 16S rRNA gene sequences has some CMVU44 . The 16S rDNA sequences of the black broom difficulties in the case of Bradyrhizobium bacteria since many nodule isolates shared 99.6–100% sequence identity. The newly described bradyrhizobial species show 99.4% or higher 16S rDNA nucleotide sequences of strains LN1, LN2, 16S rDNA sequence identity to the previously defined species LN10, LN11, LN20, LN30, and LN32 were identical to each of the genus Bradyrhizobium (Chahboune et al. 2012; other and to B. japonicum BGA-1. Interestingly, the 16S 1130 Ann Microbiol (2019) 69:1123–1134 Fig. 3 Maximum likelihood phylogenetic tree of 16S rDNA sequences of Lembotropis nigricans root nodule isolates (shown in bold) and reference bradyrhizobia. Bootstrap values ≥ 50% are given at the branching points. The scale bar indicates the number of substitutions per site. GenBank accession numbers are given in parentheses 1131 Ann Microbiol (2019) 69:1123–1134 rDNA sequence of B. japonicum BGA-1 was different from with bootstrap values of 100% and 98%. The cluster with that of B. japonicum USDA 6 at two nucleotide positions 100% confidence encompasses all black broom rhizobia and (99.8% similarity) at which it was identical to 28 reference bradyrhizobia. The B. elkanii, B. erythrophlei, B. liaoningense USDA 3622 (99.9% sequence similarity). B. valentinum, and B. lablabi species were assigned to the This observation clearly indicates a very low level of 16S other group. The same splitting of the Bradyrhizobium species rRNA gene sequence heterogeneity among Bradyrhizobium was previously described for phylogenetic trees reconstructed species. It also suggests that 16S rRNA as a molecular marker using 16S rDNA sequences (Menna et al. 2009; Delamuta has serious limitations for species delineation. The results of et al. 2012;KalitaandMałek 2017). The pattern of branching comparative 16S rDNA sequence analysis confirmed those of on the phylogram shown in Fig. 3 suggests a close relationship the numerical analysis of phenotypic features (Fig. 1) and of the studied black broom rhizobia with B. japonicum BGA- showed that the black broom rhizobia are members of the 1. However, due to the high level of 16S rRNA sequence genus Bradyrhizobium species. The phylogenetic relatedness conservation between the Bradyrhizobium species described of the L. nigricans rhizobia with other nodule bacteria is pre- above, where two strains of different species can have more sented in the form of a phylogram in Fig. 3. On the 16S rDNA similar 16S rDNA nucleotide sequences than two strains of tree, the L. nigricans symbionts and reference the same species, additional analyses were used to determine Bradyrhizobium strains were divided into two distinct clusters the species position of the L. nigricans root isolates. Fig. 4 Maximum likelihood phylogenetic tree of concatenated atpD, values ≥ 50% are given at branching points. The scale bar indicates the dnaK, gyrB,and rpoB gene sequences of Lembotropis nigricans root number of substitution per site. GenBank accession numbers are given in nodule isolates (shown in bold) and reference bradyrhizobia. Bootstrap parentheses 1132 Ann Microbiol (2019) 69:1123–1134 Analysis of concatenated atpD-dnaK-gyrB-rpoB gene multilocus sequence analysis based on four markers. sequences Nevertheless, the phylogram constructed using concatenation of three gene sequences (atpD-dnaK-rpoB) strongly supports Phylogenetic analysis was carried out using concatenated nu- the grouping of the L. nigricans isolates and B. japonicum cleotide sequences of four housekeeping genes: atpD, dnaK, BGA-1 on the 16S rRNA phylogenetic tree (Supplementary gyrB,and rpoB. Housekeeping genes have been widely used Figure S4). It also supports our conclusion that L. nigricans in many studies of Bradyrhizobium bacteria to delineate close- microsymbionts belong to B. japonicum. ly related species (Vinuesa et al. 2005; Rivas et al. 2009; All the results presented in this study clearly indicate that Chahboune et al. 2012; Delamuta et al. 2013; Kalita and L. nigricans, a plant of tribe Genisteae which has not been Małek 2017). Although the protein-encoding genes used in previously studied for bacteria inhabiting its root nodules, is the phylogenetic analysis of Bradyrhizobium bacteria display infected by strains belonging to B. japonicum species. The a considerably higher level of sequence diversity compared to present study supports our previous results and data reported 16S rDNA, no threshold value that could be used for species by other authors that root nodules of the tribe Genisteae plants demarcation has ever been proposed as it was done in case of are mainly inhabited by bacteria of the genus Bradyrhizobium ANI (average nucleotide identity), where it is widely accepted (Kalita and Małek 2010;Stępkowski et al. 2011;Kalita and that ≥ 95% ANI represents an accurate threshold for delineat- Małek 2017;Stępkowski et al. 2018). ing almost all currently named prokaryotic species (Chun et al. Funding The study was conducted by the research fund of Faculty of 2018;Jainet al. 2018). Biotechnology and Biology, Maria Curie-Skłodowska University, Lublin, The black broom rhizobia shared from 98.9 to 99.8% sim- Poland. ilarity of concatenated sequences of the four studied genes. The L. nigricans strains were most similar to B. japonicum USDA 6 (96.7 to 97.2%). The identity of the analyzed se- quences of L. nigricans bradyrhizobia to other Open Access This article is distributed under the terms of the Creative Bradyrhizobium species ranged from 87.7% in the case of Commons Attribution 4.0 International License (http:// T T B. retamae Ro19 to 95.3% for B. canariense BTA-1 .It creativecommons.org/licenses/by/4.0/), which permits unrestricted use, should be noticed that the highest similarity value of distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link L. nigricans microsymbionts to B. japonicum USDA 6 to the Creative Commons license, and indicate if changes were made. (97.2%) is lower than the sequence similarity between T T B. elkanii USDA 76 and B. pachyrhizi PAC48 (98.4%). Moreover, the lowest sequence similarity estimated between B. japonicum USDA 6 and black broom bradyrhizobia References (96.7%) overlaps the atpD-dnaK-gyrB-rpoB sequence simi- larity of B. paxllaeri LMTR21 and B. lablabi Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local CCBAU23086 (96.7%). These data indicate that there is no alignment search tool. J Mol Biol 215:403–410 clear gap for interspecific sequence similarity values since, as Aserse AA, Räsänen LA, Assefa F, Hailemariam A, Lindström K (2012) Phylogeny and genetic diversity of native rhizobia nodulating com- demonstrated above, two species can exhibit higher identity mon bean (Phaseolus vulgaris L.) in Ethiopia. Syst Appl Microbiol than strains belonging to a single species. 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Numerical analysis of phenotypic properties, genomic fingerprinting, and multilocus sequence analysis of Bradyrhizobium strains isolated from root nodules of Lembotropis nigricans of the tribe Genisteae

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Springer Journals
Copyright
Copyright © 2019 by The Author(s)
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
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1869-2044
DOI
10.1007/s13213-019-01491-6
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Abstract

Purpose The aim of this study was to estimate the level of genomic and phenotypic diversity as well as the genus and species position of bacterial strains isolated from root nodules of Lembotropis nigricans (family Fabaceae). Methods The genomic diversity of studied L. nigricans nodule symbionts was examined by using BOX-PCR and AFLP (amplified fragment length polymorphism) fingerprinting techniques. To assign bacteria to the genus, numerical analysis of phenotypic features and comparative analysis of 16S rDNA sequences were performed. The comparative analysis of combined atpD, dnaK, gyrB,and rpoB gene sequences (multilocus sequence analysis, MLSA) was used to determine the most closely related species to the studied bacteria. Results Both BOX-PCR and AFLP techniques revealed a high level of genomic heterogeneity of L. nigricans nodulators. Among 33 studied bacteria, 32 genotypes were delineated by the AFLP method and 27 genotypes were identified by the BOX-PCR fingerprinting. The numerical analysis of 86 phenotypic characteristics of L. nigricans nodule isolates and reference rhizobia showed that studied bacteria belong to the genus Bradyrhizobium.Affiliation of L. nigricans nodule isolates to the genus Bradyrhizobium was supported by comparative analysis of 16S rDNA sequences and the concatenation of atpD, dnaK, gyrB,and rpoB gene sequences. MLSA indicated also that L. nigricans microsymbionts are members of Bradyrhizobium japonicum. Conclusion L. nigricans root nodule symbionts are members of Bradyrhizobium japonicum and exhibit high phenotypic and genomic diversity important for their survival in soil. . . . . . Keywords AFLP BOX-PCR Bradyrhizobium Genisteae Lembotropis nigricans Multilocus sequence analysis Introduction Rhizobia are soil-inhabiting bacteria capable of establishing nitrogen-fixing symbiosis with plant species of the family Fabaceae. Genisteae encompassing more than 600 species Magdalena Wójcik and Michał Kalita contributed equally to this work. distributed among 25 genera is one of the largest tribes within The GenBank accession numbers for sequences generated in this study thelegumefamily (Cardoso et al. 2013). There are published are as follows: MK183761-MK183771 (16S rRNA), MK202810- reports on the phylogeny and taxonomy of bacterial strains MK202820 (atpD), MK202821-MK202831 (dnaK), MK202832- isolated from root nodules of 77 plant species of the tribe MK202842 (gyrB), MK202843-MK202853 (rpoB). Genisteae. Based on these reports, it was concluded that the Electronic supplementary material The online version of this article strains of the genus Bradyrhizobium form a predominant (https://doi.org/10.1007/s13213-019-01491-6) contains supplementary group infecting Genisteae legumes (Stępkowski et al. 2018). material, which is available to authorized users. Still, there are no data available on rhizobia of 14 Genisteae genera. One of such plant genus is Lembotropis comprising * Michał Kalita michal.kalita@umcs.pl two species: Lembotropis nigricans (L. Griseb.) and Lembotropis emeriflora (Rchb.). L. nigricans (black broom) is distributed in Central and Southeastern Europe; however, its Department of Genetics and Microbiology, Maria Curie Sklodowska University, Akademicka 19, 20-033 Lublin, Poland exact range is hard to estimate since it has been grown as an 1124 Ann Microbiol (2019) 69:1123–1134 ornamental shrub for many years and it can currently be found were used for root nodule collection. The rhizobia studied in many areas as a wild plant (Petrowicz 1981). Although it were isolated from root nodules with the standard procedure has been previously demonstrated that L. nigricans produces (Kalita and Małek 2004). Yeast extract mannitol medium cylindrical root nodules (Łotocka et al. 2012), the bacteria (YEM) was routinely used for cultivation of the rhizobia at residing in these structures have not been analyzed for their 28 °C. The strains were stored as source cultures on YEM phenotypic properties, genomic diversity, and taxonomic slants at 4 °C. affiliation. Polyphasic taxonomy, which integrates all available geno- Phenotypic characterization and numerical analysis typic, phenotypic, and phylogenetic information, has been of phenotypic properties widely used for identification and classification of bacteria since the 1970s (Vandamme and Peeters 2014). Numerical L. nigricans nodule isolates were examined for cell morphol- analysis of phenotypic and genotypic features allows differen- ogy, motility, and generation time according to a procedure tiation of closely related bacteria and preliminary determina- described earlier by Wdowiak and Małek (2000). Their phe- tion of the genus position of microorganisms. The PCR fin- notypic properties, i.e., temperature and pH growth ranges, gerprinting techniques such as BOX-PCR and AFLP are often NaCl tolerance, acid and alkali production in YEM agar sup- used for the assessment of the genomic diversity of rhizobia plemented with bromothymol blue as a pH indicator, utiliza- isolated from root nodules of different legume species (Kalita tion of different compounds as sole carbon and nitrogen and Małek 2006; Liu et al. 2011; Gnatetal. 2015;Xu et al. sources, resistance to antibiotics and dyes, reaction in litmus 2016; Wdowiak-Wróbel et al. 2017; Chidebe et al. 2018). milk, activity of urease, catalase, β-galactosidase, phospha- Since the 1990s, the phylogenetic studies of root nodule tase, nitrate reductase, cytochrome oxidase, β-D-glucosidase, isolates have been based mainly on partial or complete 16S and peroxidase, synthesis of indole, melanin, and indole-3- rDNA sequences (Young et al. 1991; Vinuesa et al. 1998). acetic acid (IAA), adsorption of Congo red and Calcofluor, Since then, many studies have demonstrated that the diversity and precipitation of calcium glycerophosphate were deter- in the 16S rRNA sequences of rhizobia is low and distin- mined according to Wdowiak and Małek (2000). The utiliza- guishes species poorly (Menna et al. 2009;Delamutaet al. tion of various compounds as a sole carbon source was studied 2013). Due to the limitations of the 16S rRNA gene analysis, in modified Bergersen’s synthetic medium (BS medium) in protein-encoding genes with evolution rates faster than that of which mannitol was replaced by the tested compound. The 16S rDNA, but conserved sufficiently to retain genetic infor- utilization of various compounds as a sole nitrogen source mation, have been proposed as alternative phylogenetic was examined in the same BS medium with the tested sub- markers. atpD, dnaK, glnII, gyrB, recA,and rpoB are exam- stance instead of NH Cl. All phenotypic tests were done in ples of housekeeping genes that are frequently used in tracing triplicate and repeated two times. The results were scored after the evolutionary history of bacteria (Gaunt et al. 2001; 5 days for fast-growing strains and 7–10 days for slow- Stępkowski et al. 2003;Vinuesa etal. 2005; Rivas et al. growing strains (Garrity et al. 2005). 2009; Kalita and Małek 2017; Huang et al. 2018). For numerical analysis, the phenotypic features of the In this study, we examined the genus position and evolu- rhizobia studied were coded in the binary system. Next, the tionary history of L. nigricans root nodule isolates using nu- simple matching similarity coefficient (SM) of each strain pair merical analysis of phenotypic properties and comparative was estimated and a similarity matrix was generated (Sneath sequence analysis of 16S rRNA, atpD, dnaK, gyrB,and and Sokal 1973). Based on the similarity matrix, clustering rpoB genes. L. nigricans symbionts were also examined for analysis was performed with the unweighted pair group meth- BOX-PCR and AFLP patterns to study their genomic relation- od with arithmetic mean (UPGMA) using the NTSYSpc soft- ship and diversity. ware package (Exeter Software). Isolation of total genomic DNA Materials and methods For DNA isolation, bacteria were grown in 30 ml of liquid Bacterial strains YEM medium for 4–5 days at 28 °C. DNA was extracted and purified according to the method proposed by Pitcher et al. The strains used in this study and their origin are listed in (1989) as described elsewhere (Kalita and Małek 2004). Supplementary Table S1.Rootnodules of L. nigricans were collected from plants growing at a single location in southeast BOX-PCR, AFLP, and data analysis Poland (51° 08′ 47.2″ N23° 24′ 43.3″ E). The L. nigricans population occupied area of approximately 50 m . Ten ran- The primers and PCR cycling conditions used in the BOX- domly selected individual plants from five distinct patches PCR and AFLP analyses are listed in Supplementary 1125 Ann Microbiol (2019) 69:1123–1134 Table S2. The AFLP procedure was done as described by Results and discussion Tyrka (2002) with some small modifications as described else- where (Kalita and Małek 2006). Each reaction was repeated Phenotypic properties of L. nigricans rhizobia three times on the same DNA matrix. Only informative and reproducible products of the genomic fingerprinting reactions A total of 48 bacterial strains, including 33 L. nigricans were analyzed. The PCR products were separated in 1.5% symbionts and 15 reference strains, representing different agarose gel, stained with ethidium bromide, and visualized species of the genera: Bradyrhizobium, Ensifer, under UV light. The DNA profiles yielded by the BOX- Mesorhizobium,and Rhizobium were analyzed for 86 phe- PCR and AFLP methods were analyzed using BIO-GEN pro- notypic properties. Table 1 presents some physiological gram version 11.01 (Vilber-Lourmat). The strains were and metabolic properties of the tested bacteria. In grouped by the Nei and Li coefficient (Nei and Li 1979)and Supplementary Table S3, the results of all 86 characteris- the dendrogram was constructed using the UPGMA method. tics are shown. The growth temperature range for the black broom rhizobia was determined to be between 13 and 3 °C Amplification and sequencing of 16S rRNA, atpD, with an optimal temperature of 28–30 °C, typical for most dnaK, gyrB,and rpoB genes rhizobia. All L. nigricans symbionts, likewise the reference Bradyrhizobium species, grew at pH 5–8. No strain toler- The primers and PCR amplification conditions used in this ated pH 4. Forty-five percent of the strains were able to study are listed in Supplementary Table S2. All PCR amplifi- grow at pH 9 and 21% grew even at pH 10. The black cation reactions were carried out with ReadyMix™ Taq PCR broom rhizobia were able to grow on YEM medium with Reaction Mix (Sigma) according to the manufacturer’srecom- 0.5 and 1% NaCl. Most of them tolerated 2% sodium chlo- mendations. DNA-free water was used in negative PCR con- ride and 32% of the studied isolates tolerated 3% NaCl, in trols. Genomic DNA of B. japonicum USDA6 wasusedasa contrast to the genus Bradyrhizobium strains, which gen- template in positive PCR controls. The amplified products erally exhibit high sensitivity to salinity (Garrity et al. were purified with Clean-Up or Gel-Out purification columns 2005). The L. nigricans symbionts were slow-growing (A&A Biotechnology) and sequenced with the BigDye rhizobia with generationtime~6hinYEM brothat Terminator Cycle sequencing kit (Thermo Fisher Scientific) 28 °C. Such a doubling time is characteristic for the strains using the 3500 Genetic Analyzer according to the manufac- of the genus Bradyrhizobium (van Berkum and Eardly turer’s procedures. 1998). Tests for assimilation of different compounds as sole carbon and nitrogen sources are commonly used in Phylogenetic analysis taxonomic studies of rhizobia. In our experiments, 25 dif- ferent carbon-containing compounds were tested as sole The sequences yielded in this study were compared to the sources of carbon for bacteria. All the tested isolates uti- nucleotide sequences from GenBank database using the lized 12 of the 25 studied carbon sources, i.e., L-alanine, L- BLASTprogram (Altschuletal. 1990). Since all the se- arginine, dextrin, mannitol, glycerol, sucrose, Tween-20, quences were most similar to the sequences of the genus D-fructose, D-galactose, insulin, D-xylose, and sodium tar- Bradyrhizobium strains, only reference bradyrhizobia were trate. The other carbon compounds served as growth sub- included for further phylogenetic analyses. All phylogenetic strates for only some bacteria studied (Table 1). In YEM analyses were conducted in MEGA 7 (Kumar et al. 2016)as medium with mannitol as a sole carbon source, black follows. Multiple sequence alignments were constructed and broom symbionts synthesized acid products similar to the resulting alignments were corrected manually. To obtain Rhizobium, Ensifer,and Mesorhizobium species strains the same number of analyzed positions in the alignment, lon- (Garrity et al. 2005;Gnatetal. 2014). Most of the tested ger sequences were truncated. As a result, 1228, 429, 204, strains did not utilize disaccharides such as maltose and 563, and 452 nucleotide positions were analyzed for the 16S trehalose similar to slow-growing bradyrhizobia, which rRNA, atpD, dnaK, gyrB,and rpoB genes, respectively. The generally do not have disaccharide uptake systems maximum likelihood (ML) method was used to reconstruct (Glenn and Dilworth 1981;Elkan 1992). However, most the phylogeny of the analyzed genes. jModelTest (Darriba of them exhibited good growth on lactose and sucrose. et al. 2012) was used to choose the best-fit evolutionary model Of the 21 nitrogen sources tested, only DL-ornithine was for each studied gene. To determine the degree of the statisti- not utilized by all the L. nigricans symbionts, whereas 81 cal support for the branches in the phylogeny, 1000 bootstrap and 79% of the bacteria did not utilize L-lysine and sodium replicates of the data were analyzed. Sequence identity values hippurate, respectively. The relevant phenotypic traits that for atpD-dnaK-gyrB-rpoB concatenation were calculated differentiated the L. nigricans nodulators and the reference using BioEdit software (Hall 2011) based on the multiple Bradyrhizobium species are listed in Table 1. An important alignment constructed in MEGA7. taxonomic criterion used in differentiation of fast-growing 1126 Ann Microbiol (2019) 69:1123–1134 Table 1 Some phenotypic characteristics of Lembotropis nigricans root nodule isolates and reference Bradyrhizobium strains Characteristics Lembotropis Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium nigricans elkanii USDA liaoningense USDA yuanmingense CCBAU diazoefficiens USDA japonicum USDA a T T T T T isolates (n =33) 76 (n =1) 3622 (n =1) 10071 (n =1) 110 (n =1) 6 (n =1) Carbon sources used b c D-Arabinose + (24) ++ + + + D-Cellobiose + (21) + + + + + D-Glucose + (27) + + + + + D-Raffinose + (9) + + + + + D-Trehalose + (4) – ++ –– D-Xylose + (33) + –– ++ Dextrin + (33) –– ++ + Inulin + (33) + –– ++ Lactose + (27) –– – ++ L-Alanine + (33) + + – ++ L-Arginine + (33) + –– ++ L-Asparagine + (24) + –– ++ L-Glutamine + (29) + + + + + L-Lysine + (14) –– – – – L-Rhamnose + (14) + + + + + L-Tyrosine + (29) + –– – – Maltose + (9) – + – ++ Salicin + (21) + – ++ + Sodium citrate + (24) + –– ++ Sodium +(4) –– – – – hippurate Starch + (24) –– – – – Nitrogen sources used DL-Isoleucine + (30) + – + – + L-Glutamic acid + (33) + + – ++ L-Lysine + (6) + – + – + L-Phenylalanine + (29) + –– ++ L-Serine + (30) + –– ++ Sodium + (7) + –– – – hippurate Sodium nitrate + (31) + + + + + Resistant to pH 9.0 + (15) –– – – – pH 10.0 + (7) –– – – – 1.0% NaCl + (32) + –– – – 3.0% NaCl + (11) –– – – – Auramine +(23) + – ++ + −1 0.05 μgml Crystal violet +(2) –– – ++ −1 0.1 μgml Methyl green +(5) –– – – – −1 0.13 μgml Neutral red + (24) – ++ + + −1 0.2 μgml Ampicillin +(22) + –– ++ −1 100 μgml Ampicillin +(13) + –– ++ −1 200 μgml Rifampicin +(28) + + + + + −1 20 μgml Ann Microbiol (2019) 69:1123–1134 1127 Table 1 (continued) Characteristics Lembotropis Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium Bradyrhizobium nigricans elkanii USDA liaoningense USDA yuanmingense CCBAU diazoefficiens USDA japonicum USDA a T T T T T isolates (n =33) 76 (n =1) 3622 (n =1) 10071 (n =1) 110 (n =1) 6 (n =1) Rifampicin + (12) –– – ++ −1 200 μgml Streptomycin +(31) + + + + + −1 10 μgml Streptomycin +(13) + –– – – −1 200 μgml Tetracycline +(30) + + + + + −1 10 μgml Tetracycline + (3) + –– – – −1 40 μgml n, the number of studied strains +, −, strains were positive, negative, respectively Value in parentheses is the number of strains with positive reaction from slow-growing rhizobia is their tolerance to antibiotics BOX-PCR and AFLP analysis (Elkan 1992). The black broom nodule isolates showed rather high tolerance to the tested antibiotics, similar to In recent years, many methods have been developed for mo- the Bradyrhizobium species (Kalita and Małek 2004). lecular typing and assessing genomic heterogeneity of bacte- −1 Most of them were resistant to ampicillin (100 μgml ), ria mainly within species or within closely related species. −1 −1 rifampicin (20 μgml ), streptomycin (10 μgml ), and One of the best known genome fingerprinting techniques −1 tetracycline (10 μgml ). All of them were able to grow in based on polymerase chain reaction (PCR) is BOX-PCR of the medium supplemented with acridine orange (0.013%), highly conserved repetitive DNA sequences occurring natu- crystal violet (0.013%), methyl red (0.05%), methyl green rally in bacterial genomes (Lupski and Weinstock 1992; (0.065%), neutral red (0.1%), nigrosine (0.5%), and safra- Versalovic et al. 1994) as well as AFLP based on nucleotide nin (0.2%). Rhizobia specific to L. nigricans were highly changes within restriction sites and adjacent bases, which homogeneous in enzyme activities (except catalase and serve for primer annealing in the PCR reaction (Blears et al. phosphatase) and all of them hydrolyzed urea, reduced ni- 1998; Savelkoul et al. 1999). Both techniques generating trate, produced active cytochrome oxidase, and exhibited DNA profiles specific to a given genome were used for anal- evident alkaline reaction in the litmus milk test. ysis of the genomic diversity and genomic relationship of the The phenotypic properties of the L. nigricans rhizobia and 33 L. nigricans root nodule isolates. In the BOXA1R-PCR the reference strains representing the genera method, a 22-base oligonucleotide primer containing 68% of Bradyrhizobium, Ensifer, Mesorhizobium,and Rhizobium GC was applied. The use of the GC-rich primer in the BOX- were subjected to numerical analysis with the use of the PCR method was associated with the high GC content of NTSYSpc software. The resulting dendrogram is shown in rhizobial genomes (Garrity et al. 2005) and helped to maxi- Fig. 1. The complete binary matrix table used for the numer- mize the number of amplicons. In the PCR reaction with all ical analysis is available as Supplementary Table 3. On the black broom rhizobia, the BOXA1R primer produced DNA basis of the cluster analysis, all strains included into analysis bands ranging in the size from 275 to 2134 nucleotides, with formed two phena at the similarity coefficient level of 0.71. an average number of nine bands per strain. The DNA BOX Phenon I included bacteria of the genera Rhizobium, Ensifer, patterns were used for cluster analysis and presentation of the and Mesorhizobium. Phenon II contained slow-growing genomic relationship in the form of a dendrogram (Fig. 2). In strains of the genus Bradyrhizobium and L. nigricans sym- the generated dendrogram, the 33 L. nigricans symbionts were bionts, which formed a separate subgroup at a similarity divided into three main clusters. In the first cluster, two strains coefficient of 0.82. The results presented in the dendrogram (LN1 and LN14) were placed distinctly from the other bacte- indicate that the slow-growing black broom rhizobia belong ria. The second main cluster comprised 17 isolates with a to the genus Bradyrhizobium. The high level of phenotypic DNA pattern similarity level from 71 to 100%, whereas the diversity of the L. nigricans symbionts determined by the third one encompassed 14 strains with a DNA pattern similar- numerical analysis may facilitate their survival in changing ity level in the range from 82 to 100%. The BOX-PCR method environmental conditions in which these bacteria naturally allowed identification of 27 genomotypes among the 33 black live. broom microsymbionts. Eleven strains belong to 5 clusters 1128 Ann Microbiol (2019) 69:1123–1134 Fig. 1 UPGMA dendrogram showing phenotypic relationships among 33 Lembotropis nigricans root nodule isolates and reference strains comprising at least two root nodule isolates sharing identical AFLP profiles of the bacteria studied contained from 1 to 19 DNA profiles (Fig. 2). DNA bands per strain and their size was from 184 to 1486 bp. To investigate the genomic diversity of the L. nigricans On the basis of DNA banding profiles, it was possible to symbionts in greater detail, the AFLP technique was also differentiate all black broom rhizobia except two strains, used. Presently, bacteriologists frequently use this method in which exhibited identical genomic patterns in the AFLP meth- the assessment of genomic diversity both within species and od with the PsI-GC primer. The genomic relationships of across different bacterial populations (Aserse et al. 2012;Li L. nigricans symbionts based on AFLP fingerprinting data et al. 2012;Xuet al. 2016). For fingerprinting the black broom are presented in the dendrogram generated by UPGMA cluster rhizobial genomes and assessment of their genomic relation- analysis (Fig. 2). In this tree, the rhizobia studied were split ships with the AFLP method, PstI endonuclease recognizing a into two major groups at a DNA similarity coefficient level of GC-rich sequence 5′-CTGGAG-3′ and the primer pair PstI- 0.55. One cluster encompassed ten rhizobia, including two GC with GC as arbitrary bases at the 3′ end were used. The strains (LN22 and LN23) sharing identical DNA patterns. 1129 Ann Microbiol (2019) 69:1123–1134 Fig. 2 Dendrograms based on (a) BOX-PCR and (b)AFLP dataof 33 Lembotropis nigricans root nodule isolates. Nei and Li coef- ficient was used for similarity measure. UPGMA was used for clustering The other group comprised 23 strains separated into two Guerrouj et al. 2013; Grönemeyer et al. 2017; Costa et al. genomically different subgroups at a similarity coefficient lev- 2018). el of 0.62. Genome heterogeneity of rhizobia is shaped by In order to clarify the genus position of the L. nigricans environmental factors and limited by the symbiotic interaction symbionts and investigate their evolutionary relationship with with the host plant. Our studies showed that the AFLP tech- other bacteria of the genus Bradyrhizobium, the nearly full- nique was superior to the BOX-PCR method in differentiating length 16S rRNA encoding genes of 11 symbionts the L. nigricans symbionts and facilitated identification of 32 representing different phenotypic and genomic groups of genomotypes among the 33 bacterial strains studied. We also black broom nodule isolates were amplified and sequenced. showed that both these genome profiling techniques used of- The 16S rDNA sequences of the studied rhizobia were aligned fer a convenient way to choose the representative strains from and compared with those of other nodule bacteria available in each genomic group for further taxonomic studies, such as the GenBank database. The evolutionary distances between 16S rDNA and multilocus sequence analysis (MLSA). L. nigricans symbionts and reference bacteria representing the genus Bradyrhizobium were calculated from a 1228 bp- long alignment and molecular phylogeny was reconstructed 16S rDNA sequence analysis using the maximum likelihood method (ML). The level of sequence similarity between the 16S rDNA of the black Comparative analysis of 16S rDNA sequences is widely used broom rhizobia and those of Bradyrhizobium ranged from to study the taxonomic position of bacteria at the genus level 94.8 to 100%. The L. nigricans rhizobia were most similar and to depict bacterial phylogeny. It was demonstrated that a (≥ 99.5%) in their 16S rDNA sequences to B. japonicum T T 16S rRNA gene sequence similarity lower than 98.7% sug- USDA 6 , B. canariense BTA-1 , B. liaoningense USDA T T gests that bacterial strains belong to distinct species (Yarza 3622 , B. daqingense CCBAU 15774 ,and B. americanum et al. 2014). Analysis of 16S rRNA gene sequences has some CMVU44 . The 16S rDNA sequences of the black broom difficulties in the case of Bradyrhizobium bacteria since many nodule isolates shared 99.6–100% sequence identity. The newly described bradyrhizobial species show 99.4% or higher 16S rDNA nucleotide sequences of strains LN1, LN2, 16S rDNA sequence identity to the previously defined species LN10, LN11, LN20, LN30, and LN32 were identical to each of the genus Bradyrhizobium (Chahboune et al. 2012; other and to B. japonicum BGA-1. Interestingly, the 16S 1130 Ann Microbiol (2019) 69:1123–1134 Fig. 3 Maximum likelihood phylogenetic tree of 16S rDNA sequences of Lembotropis nigricans root nodule isolates (shown in bold) and reference bradyrhizobia. Bootstrap values ≥ 50% are given at the branching points. The scale bar indicates the number of substitutions per site. GenBank accession numbers are given in parentheses 1131 Ann Microbiol (2019) 69:1123–1134 rDNA sequence of B. japonicum BGA-1 was different from with bootstrap values of 100% and 98%. The cluster with that of B. japonicum USDA 6 at two nucleotide positions 100% confidence encompasses all black broom rhizobia and (99.8% similarity) at which it was identical to 28 reference bradyrhizobia. The B. elkanii, B. erythrophlei, B. liaoningense USDA 3622 (99.9% sequence similarity). B. valentinum, and B. lablabi species were assigned to the This observation clearly indicates a very low level of 16S other group. The same splitting of the Bradyrhizobium species rRNA gene sequence heterogeneity among Bradyrhizobium was previously described for phylogenetic trees reconstructed species. It also suggests that 16S rRNA as a molecular marker using 16S rDNA sequences (Menna et al. 2009; Delamuta has serious limitations for species delineation. The results of et al. 2012;KalitaandMałek 2017). The pattern of branching comparative 16S rDNA sequence analysis confirmed those of on the phylogram shown in Fig. 3 suggests a close relationship the numerical analysis of phenotypic features (Fig. 1) and of the studied black broom rhizobia with B. japonicum BGA- showed that the black broom rhizobia are members of the 1. However, due to the high level of 16S rRNA sequence genus Bradyrhizobium species. The phylogenetic relatedness conservation between the Bradyrhizobium species described of the L. nigricans rhizobia with other nodule bacteria is pre- above, where two strains of different species can have more sented in the form of a phylogram in Fig. 3. On the 16S rDNA similar 16S rDNA nucleotide sequences than two strains of tree, the L. nigricans symbionts and reference the same species, additional analyses were used to determine Bradyrhizobium strains were divided into two distinct clusters the species position of the L. nigricans root isolates. Fig. 4 Maximum likelihood phylogenetic tree of concatenated atpD, values ≥ 50% are given at branching points. The scale bar indicates the dnaK, gyrB,and rpoB gene sequences of Lembotropis nigricans root number of substitution per site. GenBank accession numbers are given in nodule isolates (shown in bold) and reference bradyrhizobia. Bootstrap parentheses 1132 Ann Microbiol (2019) 69:1123–1134 Analysis of concatenated atpD-dnaK-gyrB-rpoB gene multilocus sequence analysis based on four markers. sequences Nevertheless, the phylogram constructed using concatenation of three gene sequences (atpD-dnaK-rpoB) strongly supports Phylogenetic analysis was carried out using concatenated nu- the grouping of the L. nigricans isolates and B. japonicum cleotide sequences of four housekeeping genes: atpD, dnaK, BGA-1 on the 16S rRNA phylogenetic tree (Supplementary gyrB,and rpoB. Housekeeping genes have been widely used Figure S4). It also supports our conclusion that L. nigricans in many studies of Bradyrhizobium bacteria to delineate close- microsymbionts belong to B. japonicum. ly related species (Vinuesa et al. 2005; Rivas et al. 2009; All the results presented in this study clearly indicate that Chahboune et al. 2012; Delamuta et al. 2013; Kalita and L. nigricans, a plant of tribe Genisteae which has not been Małek 2017). Although the protein-encoding genes used in previously studied for bacteria inhabiting its root nodules, is the phylogenetic analysis of Bradyrhizobium bacteria display infected by strains belonging to B. japonicum species. The a considerably higher level of sequence diversity compared to present study supports our previous results and data reported 16S rDNA, no threshold value that could be used for species by other authors that root nodules of the tribe Genisteae plants demarcation has ever been proposed as it was done in case of are mainly inhabited by bacteria of the genus Bradyrhizobium ANI (average nucleotide identity), where it is widely accepted (Kalita and Małek 2010;Stępkowski et al. 2011;Kalita and that ≥ 95% ANI represents an accurate threshold for delineat- Małek 2017;Stępkowski et al. 2018). ing almost all currently named prokaryotic species (Chun et al. Funding The study was conducted by the research fund of Faculty of 2018;Jainet al. 2018). Biotechnology and Biology, Maria Curie-Skłodowska University, Lublin, The black broom rhizobia shared from 98.9 to 99.8% sim- Poland. ilarity of concatenated sequences of the four studied genes. The L. nigricans strains were most similar to B. japonicum USDA 6 (96.7 to 97.2%). The identity of the analyzed se- quences of L. nigricans bradyrhizobia to other Open Access This article is distributed under the terms of the Creative Bradyrhizobium species ranged from 87.7% in the case of Commons Attribution 4.0 International License (http:// T T B. retamae Ro19 to 95.3% for B. canariense BTA-1 .It creativecommons.org/licenses/by/4.0/), which permits unrestricted use, should be noticed that the highest similarity value of distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link L. nigricans microsymbionts to B. japonicum USDA 6 to the Creative Commons license, and indicate if changes were made. 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Annals of MicrobiologySpringer Journals

Published: Jun 15, 2019

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