recA Gene Sequences Research Articles

RecA gene genetic diversity and its regulatory element analysis: The case of Vibrio cholerae

In conventional systems, indicator organisms are used to monitor drinking water quality which is unspecific in detecting pathogens. On the contrary, molecular techniques play vital role in these aspects by providing precise genetic information about particular pathogen of interest via proper biomarkers. The aim of this study was to undertake genetic relatedness and promoter region analysis for V. cholerae strains based on recA gene sequences which were retrieved from NCBI. Sequences homologous were ensured using BLASTn from RefSeq sequence in the NCBI using V. cholerae serotype O1 strain N16961 as a reference. Accordingly, considering only complete and coding sequences with query coverage of greater than 90% and E-value of less than 5%, a total of 109 sequences were recovered from NCBI. The genetic diversity analysis was carried out using MEGA X. The analysis revealed more than 99% sequence similarity among V. cholerae strains. From promoter regions analyses, five motifs shared by all (100%) of the promoter input sequences were identified. Using TOMTOM web application, motif_1was compared to a known prokaryotes' transcription factor database to search for its similarity with known regulatory motifs and was shown to serve as abundant binding site for helix-turn-helix (HTH) transcription factor gene family to regulate expression of V. cholerae recA genes. Furthermore, our analysis revealed that the V. cholerae recA gene promoter regions are CpG islands rich. In conclusion, all V. cholerae strains shared a very high degree of recA gene sequence similarity apart from other vibrios, which indicates that these non-virulent strains may be the remnant of pathogenic strains with the loss of toxin encoding genes.

Phylogenetic Analyses of Rhizobia Isolated from Nodules of Lupinus angustifolius in Northern Tunisia Reveal Devosia sp. as a New Microsymbiont of Lupin Species

Thirty-two bacterial isolates were obtained from root nodules of Lupinus angustifolius growing in Northern Tunisia. Phylogenetic analyses based on recA and gyrB partial gene sequences grouped the strains into six clusters: four clusters belonged to the genus Bradyrhizobium (22 isolates), one to Microvirga (8 isolates) and one to Devosia (2 isolates), a genus that has not been previously reported to nodulate lupin. Representative strains of each group were further characterized. Multi-Locus Sequence Analysis (MLSA) based on recA and glnII gene sequences separated the strains within the genus Bradyrhizobium into four divergent clusters related to B. canariense, B. liaoningense, B. lupini, and B. algeriense, respectively. The latter might constitute a new Bradyrhizobium species. The strains in the Microvirga cluster showed high identity with M. tunisiensis. The Devosia isolates might also represent a new species within this genus. An additional phylogenetic analysis based on the symbiotic gene nodC affiliated the strains to symbiovars genistearum, mediterranense, and to a possibly new symbiovar. These results altogether contributed to the existing knowledge on the genetic diversity of lupin-nodulating microsymbionts and revealed a likely new, fast-growing, salt-tolerant rhizobial species within the genus Devosia as a potentially useful inoculant in agricultural practices or landscape restoration.

Phylogenetic relationships and antibiotic resistance of Vibrio parahaemolyticus isolates related to acute hepatopancreatic necrosis disease in Korea

In this study, we performed genotype analysis based on multi-locus sequence typing (MLST) and antibiotic resistance of 27 pirAB-harboring Vibrio parahaemolyticus (VpAHPND) isolates recovered from white leg shrimp (Penaeus vannamei) from acute hepatopancreatic necrosis disease (AHPND) affected farms (in 2016, n = 22) and retail markets (in 2016, n = 5) of Korea. Most VpAHPND isolates were resistant to colistin, ampicillin, and streptomycin but susceptible to other antibiotics. The comparison of their antibiotic resistance to that of non-VpAHPND isolates revealed a significantly higher resistance of VpAHPND isolates to streptomycin. No differences, however, were observed in the minimal inhibitory concentration median (MIC50), 90th percentile (MIC90), and average multiple antibiotic resistance (MAR) index between the two groups. In MLST analysis, sequence types could not be defined for 19 isolates owing to the failure of recA gene sequencing. Seven new STs were found in five and two isolates from retail markets and AHPND affected farms, respectively. Based on the MLST-based phylogenetic tree, all isolates were divided into eight clades; 22 isolates from AHPND affected farms were grouped into three clades, and five isolates from retail markets formed singleton clades. These data suggest that most AHPND outbreaks in Korea may be related to VpAHPND isolates originating from a common clone through the acquisition of pirAB. Further, the detection of VpAHPND from retail shrimp samples suggests that the pathogen may be widely distributed in shrimp aquaculture and/or the environment of Korea.

Characterization of Bradyrhizobium spp. Nodulating Lupinus cosentinii and L. luteus Microsymbionts in Morocco

In this work, we analyzed the diversity of the nodule-forming bacteria associated with Lupinus luteus and Lupinus cosentinii grown in the Maamora Cork oak forest acidic soils in Morocco. The phenotypic analysis showed the high diversity of the strains nodulating the two lupine's species. The strains were not tolerant to acidity or high alkalinity. They do not tolerate salinity or high temperatures either. The strains isolated from L. luteus were more tolerant to antibiotics and salinity than those isolated from L. cosentinii. The plant growth promoting (PGP) activities of our strains are modest, as among the 28 tested isolates, only six produced auxins, six produced siderophores, whereas three solubilized phosphates. Only two strains possess the three activities. The rrs gene sequences from eight representative strains selected following ARDRA and REP-PCR results revealed that they were members of the genus Bradyrhizobium. Six strains were then retained for further molecular analysis. The glnII, recA, gyrB, dnaK, and rpoB housekeeping gene sequence phylogeny showed that some strains were close to B. lupini LMG28514T whereas others may constitute new genospecies in the genus Bradyrhizobium. The strains were unable to nodulate Glycine max and Phaseolus vulgaris and effectively nodulated L. luteus, L. cosentinii, L. angustifolius, Chamaecytisus albidus, and Retama monosperma. The nodC and nodA symbiotic gene phylogenies showed that the strains are members of the genistearum symbiovar.

Decreasing the Cut-off Score Value of MALDI-ToF MS Increase the Identities of Burkholderia cepacia Complex Species.

Burkholderia cepacia complex (Bcc) comprises 24 related species genetically distinct, associated with high mortality in cystic fibrosis (CF) patients. Due to a high level of similarity among Bcc species, accurate identification has been problematic, and most conventional and automated phenotypic tests have shown low accuracy. We evaluated accuracy of MALDI-ToF MS decreasing the cut-off score value to distinguish Bcc species compared to recA gene sequencing. A total of 145 Bcc isolates were analyzed. B. vietnamiensis (41.37%), B. cenocepacia IIIA (23.44%), B. multivorans (20%), B. cenocepacia IIIB (11.03%), and B. contaminans (2.75%) among other species were identified by recA sequencing. MALDI-ToF MS identified 100% of Bcc isolates at the genus level and 53.1% at the species level. By decreasing cut-off values for ≥1.70, the correct identification at the species level increased to 74.5%. MALDI-ToF MS proved to be useful at the genus level identification, but it still requires improvements that allow more precise identification, requiring continuous updates and addition of new spectra to its database. A review of interpretative criteria is a field to be explored with a large collection of Bcc species.

Paraburkholderia spp. are the main rhizobial microsymbionts of Mimosa tenuiflora (Willd.) Poir. in soils of the Brazilian tropical dry forests (Caatinga biome)

Mimosa tenuiflora (Willd.) Poir. is widespread in southern and central American drylands, but little information is available concerning its associated rhizobia. Therefore, this study aimed to characterize M. tenuiflora rhizobia from soils of the tropical dry forests (Caatinga) in Pernambuco State, Brazil, at the molecular and symbiotic levels. Soil samples of pristine Caatinga areas in four municipalities were used to grow M. tenuiflora. First, the bacteria from root nodules were subjected to nodC/nifH gene amplification, and the bacteria positive for both genes had the 16S rRNA gene sequenced. Then, ten strains were evaluated using recA, gyrB, and nodC gene sequences, and seven of them had their symbiotic efficiency assessed. Thirty-two strains were obtained and 22 of them were nodC/nifH positive. Twenty strains clustered within Paraburkholderia and two within Rhizobium by 16S rRNA gene sequencing. The beta-rhizobia were similar to P. phenoliruptrix (12) and P. diazotrophica (8). Both alpha-rhizobia were closely related to R. miluonense. The recA + gyrB phylogenetic analysis clustered four and five strains within the P. phenoliruptrix and P. diazotrophica branches, respectively, but they were somewhat divergent to the 16S rRNA phylogeny. For Rhizobium sp. ESA 637, the recA + gyrB phylogeny clustered the strain with R. jaguaris. The nodC phylogeny indicated that ESA 626, ESA 629, and ESA 630 probably represented a new symbiovar branch. The inoculation assay showed high symbiotic efficiency for all tested strains. The results indicated high genetic diversity and efficiency of M. tenuiflora rhizobia in Brazilian drylands and included P. phenoliruptrix-like bacteria in the list of efficient beta-rhizobia in the Caatinga biome.

Genomics reveals the novel species placement of industrial contaminant isolates incorrectly identified as Burkholderia lata.

The Burkholderia cepacia complex (Bcc) is a closely related group of bacteria, composed of at least 20 different species, the accurate identification of which is essential in the context of infectious diseases. In industry, they can contaminate non-food products, including home and personal care products and cosmetics. The Bcc are problematic contaminants due to their ubiquitous presence and intrinsic antimicrobial resistance, which enables them to occasionally overcome preservation systems in non-sterile products. Burkholderia lata and Burkholderia contaminans are amongst the Bcc bacteria encountered most frequently as industrial contaminants, but their identification is not straightforward. Both species were historically established as a part of a group known collectively as taxon K, based upon analysis of the recA gene and multilocus sequence typing (MLST). Here, we deploy a straightforward genomics-based workflow for accurate Bcc classification using average nucleotide identity (ANI) and core-gene analysis. The workflow was used to examine a panel of 23 Burkholderia taxon K industrial strains, which, based on MLST, comprised 13 B. lata, 4 B. contaminans and 6 unclassified Bcc strains. Our genomic identification showed that the B. contaminans strains retained their classification, whilst the remaining strains were reclassified as Burkholderia aenigmatica sp. nov. Incorrect taxonomic identification of industrial contaminants is a problematic issue. Application and testing of our genomic workflow allowed the correct classification of 23 Bcc industrial strains, and also indicated that B. aenigmatica sp. nov. may have greater importance than B. lata as a contaminant species. Our study illustrates how the non-food manufacturing industry can harness whole-genome sequencing to better understand antimicrobial-resistant bacteria affecting their products.

Characterization of the Recombinant Glutamate Decarboxylase of Lactobacillus brevis G144 Isolated from Galchi Jeotgal, a Korean Salted and Fermented Seafood

A γ-aminobutyric acid (GABA)-producing microorganism was isolated from galchi (hairtail fish, Trichiurus lepturus) jeotgal, a Korean salted and fermented seafood. The G144 isolate produced GABA excessively when incubated in MRS broth containing monosodium glutamate (MSG, 3%, w/v). G144 was identified as Lactobacillus brevis through 16S rRNA and recA gene sequencing. gadB and gadC encoding glutamate decarboxylase (GAD) and glutamate/GABA antiporter, respectively, were cloned and gadB was located downstream of gadC. The operon structure of gadCB was confirmed by reverse transcription (RT)-polymerase chain reaction. gadB was overexpressed in Escherichia coli and recombinant GAD was purified and its size was 54.4 kDa as evidenced by SDS-PAGE results. Maximum GAD activity was observed at pH 5.0 and 40℃ and the activity was dependent on pyridoxal 5''-phophate. The Km and Vmax of GAD were 8.6 mM and 0.01 mM/min, respectively.

Studies of Phylogeny, Symbiotic Functioning and Ecological Traits of Indigenous Microsymbionts Nodulating Bambara Groundnut (Vigna subterranea L. Verdc)in Eswatini.

Rhizobial microsymbionts of grain legumes are ubiquitous in soils and exhibit a wide range of diversity with respect to colony morphology, genetic variability, biochemical characteristics, and phylogenetic relationships. This study assessed the phylogenetic positions of rhizobial microsymbionts of Bambara groundnut from Eswatini exhibiting variations in morpho-physiology, adaptive characteristics, and N2-fixing efficiency. Theisolates' ERIC-PCR profiles revealed the presence of high genetic variation among them. These test isolates also exhibited differences in pH tolerance and IAA production. Multilocus sequence analysis based on the 16S rRNA, atpD, glnII, gyrB, and recA gene sequences of representative test isolates closely aligned themto the type strains ofBradyrhizobium arachidis, B. manausense, B. guangdongense, B. elkanii, and B. pachyrhizi. However, some isolates showed a high divergence from the known reference type strains, indicating that they may represent species yet to be properly characterized and described. Functional characterization in the glasshouse revealed that most of the isolates from the contrasting Agro-ecologies of Eswatini were efficient in N2 fixation, and therefore elicited greater stomatal conductance and photosynthetic rates in the homologous Bambara groundnut. Of the 75 isolates tested, 51% were more effective than the commercial Bradyrhizobium sp.strain CB756, with relative symbiotic effectiveness ranging from 138 to 308%. The findings of this study indicated that the analysis ofhousekeeping genes and functional traits of Bambara-nodulating microsymbionts can provide a clear view for understanding and predicting rhizobial community structure across environmental gradients.

Comparison of Microbiological Characteristics and Genetic Diversity between Burkholderia cepacia Complex Isolates from Vascular Access and Other Clinical Infections.

Burkholderia cepacia complex (BCC) is a group of closely related bacteria with widespread environmental distribution. BCC bacteria are opportunistic pathogens that cause nosocomial infections in patients, especially cystic fibrosis (CF). Multilocus sequence typing (MLST) is used nowadays to differentiate species within the BCC complex. This study collected 41 BCC isolates from vascular access infections (VAIs) and other clinical infections between 2014 and 2020. We preliminarily identified bacterial isolates using standard biochemical procedures and further conducted recA gene sequencing and MLST for species identification. We determined genetic diversity indices using bioinformatics software. We studied 14 isolates retrieved from patients with VAIs and observed that Burkholderia cepacia was the predominant bacterial species, and B. contaminans followed by B. cenocepacia were mainly retrieved from patients with other infections. According to MLST data, we identified that all B. contaminans isolates belonged to ST102, while a wide variety of sequence types (STs) were found in B. cenocepacia isolates. In summary, the high diversity and easy transmission of BCC increase BCC infections, which provides insights into their potential clinical effects in non-CF infections.

Distribution of Burkholderia cepacia complex species isolated from industrial processes and contaminated products in Argentina.

Burkholderia cepacia complex (Bcc) members have clinical relevance as opportunistic pathogens in patients with cystic fibrosis and are responsible of numerous nosocomial infections. These closely related bacteria are also reported as frequent contaminants of industrial products. In this retrospective study, we use PCR and recA gene sequence analysis to identify at species level Bcc isolates recovered from massive consumption products and industrial processes in Argentina during the last 25years. The sequences obtained were also compared with recA sequences from clinical Bcc isolates deposited in GenBank database. We detected Bcc in purified water and preserved products from pharmaceutics, cosmetics, household cleaning articles, and beverages industries. B. contaminans (which is prevalent among people with cystic fibrosis in Argentina) was the most frequent Bcc species identified (42% of the Bcc isolates studied). B. cepacia (10%), B. cenocepacia (5%), B. vietnamiensis (16%), B. arboris (3%), and the recently defined B. aenigmatica (24%) were also detected. Rec A sequences from all B. cepacia and most B. contaminans industrial isolates obtained in this study displayed 100% identity with recA sequences from isolates infecting Argentinean patients. This information brings evidence for considering industrial massive consumption products as a potential source of Bcc infections. In addition, identification at species level in industrial microbiological laboratories is necessary for a better epidemiological surveillance. Particularly in Argentina, more studies are required in order to reveal the role of these products in the acquisition of B. contaminans infections.

Identification of Clinically Relevant Streptococcus and Enterococcus Species Based on Biochemical Methods and 16S rRNA, sodA, tuf, rpoB, and recA Gene Sequencing.

Streptococci and enterococci are significant opportunistic pathogens in epidemiology and infectious medicine. High genetic and taxonomic similarities and several reclassifications within genera are the most challenging in species identification. The aim of this study was to identify Streptococcus and Enterococcus species using genetic and phenotypic methods and to determine the most discriminatory identification method. Thirty strains recovered from clinical samples representing 15 streptococcal species, five enterococcal species, and four nonstreptococcal species were subjected to bacterial identification by the Vitek® 2 system and Sanger-based sequencing methods targeting the 16S rRNA, sodA, tuf, rpoB, and recA genes. Phenotypic methods allowed the identification of 10 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains (Leuconostoc, Granulicatella, and Globicatella genera). The combination of sequencing methods allowed the identification of 21 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains. The 16S rRNA and rpoB genes had the highest identification potential. Only a combination of several molecular methods was sufficient for unambiguous confirmation of species identity. This study will be useful for comparison of several identification methods, both those used as a first choice in routine microbiology and those used for final confirmation.

Paraburkholderia lycopersici sp. nov., a nitrogen-fixing species isolated from rhizoplane of Lycopersicon esculentum Mill. var. Saladette in Mexico

A survey of our in-house bacterial collection identified a group of six strains isolated from the tomato rhizoplane that possessed 16S rRNA gene sequences with 98.2% sequence similarity to Paraburkholderia pallida, suggesting that these strains represented a novel species. Multilocus sequence analysis using gltB, lepA and recA gene sequences showed the clustering of the strains and the BOX-PCR patterns were similar among these strains. The average nucleotide identity and the DNA-DNA virtual hybridization of strain TNe-862T was <89% and <34%, respectively, to the genomes of any sequenced Paraburkholderia species. The genome of strain TNe-862T possessed all the genes necessary for nitrogen fixation and biosynthesis of indoleacetic acid and antimicrobials terpenes, phosphonates and bacteriocins. It also contained genes for metal resistance, xenobiotic degradation, and hydrolytic enzymes such as a putative chitinase and isoamylase. Even though the strain contained potential genes for degradation of cellulose and starch, the bacterium was unable to utilize these substrates in culture medium. The genome encoded flagella and pili as well as multiple chemotaxis systems. In addition, genes encoding for the type I, II, IV, V and VI secretion systems were also present. The strains grow up to 42°C and 5% NaCl. The optimum growth pH was 8. The major cellular fatty acids were C16:0 and C18:1 ω7c. Based on this polyphasic analysis, these strains represent a novel species in the genus Paraburkholderia, for which the name Paraburkholderia lycopersici sp. nov. is proposed. The type strain is TNe-862T (=LMG 26415T=CIP 110323T).

Rhizobium rhizophilum sp. nov., an indole acetic acid-producing bacterium isolated from rape (Brassica napus L.) rhizosphere soil.

A novel Gram-stain-negative, aerobic, rod-shaped and indole acetic acid-producing strain, designated 7209-2T, was isolated from rhizosphere of rape (Brassica napus L.) grown in the Yakeshi City, Inner Mongolia, PR China. The 16S rRNA gene sequence analysis indicated that strain 7209-2T belongs to the genus Rhizobium and is closely related to Rhizobium rosettiformans W3T, Rhizobium ipomoeae shin9-1T and Rhizobium wuzhouense W44T with sequence similarities of 98.2, 98.1 and 97.9 %, respectively. Phylogenetic analysis based on concatenated housekeeping recA and atpD gene sequences showed that strain 7209-2T formed a group together with R. wuzhouense W44T and R. rosettiformans W3T, with sequences similarities of 92.6 and 91.1 %, respectively. The genome size of strain 7209-2T was 5.25 Mb, comprising 5027 predicted genes with a DNA G+C content of 61.2 mol%. The average nucleotide identity and digital DNA-DNA hybridization comparisons among 7209-2T and reference strains for the most closely related species showed values below the accepted threshold for species discrimination. The major fatty acids of strain 7209-2T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 2 (C12 : 0 aldehyde and/or unknown 10.953) . The major polar lipids were found to consist of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as quinone 10. Based on all the above results, strain 7209-2T represents a novel species of the genus Rhizobium, for which the name Rhizobium rhizophilum sp. nov. is proposed with 7209-2T (=CGMCC 1.15691T=DSM 103161T) as the type strain.

Identification and characterization of Dickeya zeae strains associated with maize stalk soft-rot in northern Serbia

Bacterial stalk soft rot have been repeatedly observed on maize plants in several commercial fields in northern part of Serbia in the period of 1990 to 2014. The occurrence of the disease corresponded with warm weather and increased humidity. Etiological studies of the diseased tissue constantly resulted in isolation of pectolytic bacterial strains. In order to identify the isolated bacteria, twenty-three strains were characterized by morphological, physiolcal, biochemical, and molecular assays. Pathogenicity of the strains was confirmed by Koch’s postulates on 1-week-old maize seedlings. The strains were Gram and oxidase-negative, non-fluorescent, pectolytic, facultative anaerobic and caused hypersensitive response (HR) in tobacco leaves. They produced catalase and lecithinase, but did not produce oxidase and arginine dehydrolase. All strains reduced nitrate and grew at 37 °C, while variable growth was observed in medium containing 5% NaCl. Phenotypic tests and amplification of the specific 420-bp fragment in PCR assay showed that the strains belong to genus Dickeya. Using ERIC-PCR analysis seven different genetic profiles were obtained, suggesting the presence of genetic diversity in the population of this pathogen in Serbia. Additionally, phylogenetic analysis based on the recA gene sequence analysis indicated that the strains isolated from soft rotted maize belong to Dickeya zeae, leading to the conclusion that this bacterium was the causal agent of stalk soft rot of maize in Serbia.

Proposal of Lactobacillus kosoi Chiou et al. 2018 as a later heterotypic synonym of Lactobacillus micheneri McFrederick et al. 2018, elevation of Lactobacillus plantarum subsp. argentoratensis to the species level as Lactobacillus argentoratensis sp. nov., and Lactobacillus zhaodongensis sp. nov., isolated from traditional Chinese pickle and the intestinal tract of a honey bee (Apis mellifera).

Lactobacillus kosoi Chiou et al. 2018 and Lactobacillus micheneri McFrederick et al. 2018 are closely related, and they share 100 % 16S rRNA gene sequence similarity, 99.6 % pheS gene sequence similarity, 100 % rpoA gene sequence similarity, 97.3 % average nucleotide identity (ANI) value and 76.6 % in silico DNA-DNA hybridization (isDDH) value, indicating that they represent the same species. Fatty acid methyl esters (FAME) analysis and phenotypic characterization also indicated that L. kosoi and L. micheneri are very similar. We propose L. kosoi Chiou et al. 2018 as a later heterotypic synonym of L. micheneri McFrederick et al. 2018. The taxonomic position of Lactobacillus plantarum subsp. argentoratensis in the L. plantarum group was re-examined using a polyphasic approach, including sequence analyses of 16S rRNA, pheS, rpoA and recA genes, average nucleotide identity analysis, in silico DNA-DNA hybridization, fatty acid methyl ester analysis and phenotypic characterization. Results of 16S rRNA gene sequence analysis indicated that L. plantarum subsp. argentoratensis was closely related to L. plantarum subsp. plantarum, L. pentosus and L. paraplantarum in the L. plantarum group, sharing 99.6-99.7 % 16S rRNA gene sequence similarities. Results of pheS, rpoA and recA gene sequence analyses indicated that L. plantarum subsp. argentoratensis was most closely related to L. plantarum subsp. plantarum, having 91.8 % pheS gene sequence similarity, 98.9 % rpoA gene sequence similarity and 93.1 % recA gene sequence similarity. L. plantarum subsp. argentoratensis DSM 16365T shared 95.6 % ANI value and 62.9 % isDDH value with L. plantarum subsp. plantarum ATCC 14917T. The low isDDH value confirmed that L. plantarum subsp. argentoratensis and L. plantarum subsp. plantarum represent two different species, rather than two different subspecies in the L. plantarum group. On the basis of the data from polyphasic characterization obtained in the present study and in previous studies, L. plantarum subsp. argentoratensis is elevated to the species level and represents a novel species of the genus Lactobacillus, for which the name Lactobacillus argentoratensis sp. nov. is proposed and the type strain is DKO 22T (=CIP 108320T=DSM 16365T=JCM 16169T). Two novel Gram-stain-positive bacterial strains, designated 1206-1T and F027-1-2, were isolated from traditional pickle in Heilongjiang Province, PR China, and from the intestinal tract of a honey bee (Apis mellifera) in Hubei Province, PR China, respectively. The two bacteria were characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, fatty acid methyl ester analysis, average nucleotide identity analysis, in silico DNA-DNA hybridization analysis and an analysis of phenotypic features. The results of 16S rRNA gene sequence analysis indicated that strains 1206-1T and F027-1-2 were distantly related to Lactobacillus sharpeae, Lactobacillus hulanensis, Lactobacillus songhuajiangensis, Lactobacillus pantheris, Lactobacillus thailandensis, Lactobacillus camelliae, Lactobacillus jixianensis, Lactobacillus nasuensis, Lactobacillus baoqingensis, Lactobacillus manihotivorans and Lactobacillus porcinae. Strain 1206-1T exhibited 94.2-96.4 % 16S rRNA gene sequence similarities, 69.5-83.3 % pheS gene sequence similarities and 73.1-90.3 % rpoA gene sequence similarities to type strains of phylogenetically related species. ANI and isDDH values between strain 1206-1T and the type strains of phylogenetically related species were 52.7-73.7 % and 21.1-30.1 %, respectively. On the basis of the data obtained in the present study, a novel species, Lactobacillus zhaodongensis sp. nov. is proposed and the type strain is 1206-1T (=CCM 8981T=CCTCC AB 2019200T=LMG 31620T).

Streptobacillus canis sp. nov. isolated from a dog.

From a phlegmon in a dog an aerobic and facultatively anaerobic, indole-, oxidase- and catalase-negative, non-motile bacterium was isolated in 2019 in Germany that stained Gram-negative and showed a pleomorphic, rod-shaped, non-spore-forming appearance. Based on the results of 16S rRNA gene sequence analyses, strain IHIT1603-19T was assigned to the genus Streptobacillus with sequence similarities of 98.6, 98.0, 97.9, 97.1 and 94.4 % to the type strains of Streptobacillus felis, Streptobacillus notomytis, Streptobacillus ratti, Streptobacillus moniliformis and Streptobacillus hongkongensis, respectively. Strain IHIT1603-19T could also clearly be differentiated from other Streptobacillus species by rpoB, groEL and recA gene, nucleotide and amino acid sequence analyses as well as by core genome phylogeny. Regarding DNA-DNA relatedness, strain IHIT1603-19T demonstrated an average nucleotide identity of 83.00 and 82.28 % compared to S. felis 131000547T and S. moniliformis DSM 12112T, respectively. Chemotaxonomic and physiological data of strain IHIT1603-19T were in congruence with other closely related members of the family Leptotrichiaceae, represented by highly similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis also proved suitable in unequivocally discriminating strain IHIT1603-19T from all currently described taxa of the genus Streptobacillus. On the basis of these data, we propose the novel species Streptobacillus canis sp. nov. with the type strain IHIT1603-19T (=DSM 110501T=CCUG 74118T=CIP 111795T). The G+C content of the DNA of the type strain is 26.6 mol%, genome size is 1.60 Mbp.

Bradyrhizobia associated with Laburnum anagyroides, an exotic legume grown in Poland

We isolated 18 rhizobial strains from root nodules of a leguminous shrub Laburnum anagyroides (common laburnum) grown in Southeast Poland as an exotic plant. With the use of BOX-PCR fingerprinting, the isolates were clustered into 2 main groups and one separate lineage, which was congruent with the ITS-RFLP results. The phylogenetic trees constructed based on 16S rRNA and combined atpD, dnaK, glnA, and recA gene sequence data separated the representative strains into three evolutionary lineages within the Bradyrhizobium jicamae supergroup, with Bradyrhizobium algeriense and Bradyrhizobium valentinum as the closest relatives. The nodA and nifH gene phylogenies proved that the L. anagyroides symbionts carry a symbiotic gene variant known as Clade IV, representing the symbiovar retamae. Phenotypic characteristics of the isolates and reference strains are also reported. Our study of the rhizobia nodulating L. anagyroides growing in Poland complements earlier few findings on the symbiotic associations of this Genisteae species.

Weissella sagaensis sp. nov., isolated from traditional Chinese yogurt.

Three Gram-stain-positive bacterial strains, designated X0750T, X0278 and X0401, isolated from traditional yogurt in Tibet Autonomous Region, PR China, were characterized by a polyphasic approach, including sequence analyses of the 16S rRNA gene and three housekeeping genes (pheS, rpoA and recA), determination of average nucleotide identity (ANI) and average amino acid identity (AAI), in silico DNA-DNA hybridization (isDDH), fatty acid methyl ester (FAME) analysis and phenotypic characterization. Strain X0750T was phylogenetically related to the type strains of Weissella hellenica, Weissella bombi, Weissella paramesenteroides, Weissella jogaejeotgali, Weissella thailandensis, Weissella oryzae, Weissella cibaria and Weissella confusa, having 94.4-100 % 16S rRNA gene sequence similarities, 76.7-90.0 % pheS gene sequence similarities, 88.9-99.4 % rpoA gene sequence similarities and 77.6-92.8 % recA gene sequence similarities, respectively. ANI, isDDH and AAI values between strain X0750T and type strains of phylogenetically related species were less than 90.4, 40.9 and 92.8 % respectively, confirming that strain X0750T represents a novel species within the genus Weissella. Based upon the data obtained in the present study, a novel species, Weissella sagaensis sp. nov., is proposed and the type strain is X0750T(=NCIMB 15192T=CCM 8924T=LMG 31184T=CCTCC AB 2018403T).

Genetically diverse lentil- and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia.

In total 196 bacterial isolates were obtained from root nodules of lentil (Lens culinaris) and faba bean (Vicia faba) grown on soil samples collected from 10 different sites in central and southern parts of Ethiopia. All isolates were identified as members of the genus Rhizobium by using recA gene sequence analysis. In the recA phylogenetic tree 195 rhizobial strains were classified into nine genospecies. The phylogeny of symbiotic genes nodC and nifH revealed five and six distinct groups respectively, largely dominated by symbiovar viciae. A multivariate analysis showed that environmental variables of the sampling sites considered in this study had more effect on the distribution and composition of the genospecies than the host legumes of the strains. Twenty representative strains, selected based on their isolation site, host plant and nodC group, were able to nodulate all lentil, faba bean, field pea (Pisum abyssinicum) and grass pea (Lathyrus sativus) plants in a greenhouse test in axenic conditions. The majority of the rhizobial strains were effective nitrogen-fixing symbionts for all tested legumes, indicating their potential to serve as broad host-range inoculants in agriculture. The present work suggests the presence of taxonomically and symbiotically diverse rhizobial species for legumes in the Viciae tribe in Ethiopia.