Novel nitrogen-fixing bacteria isolated from the root nodules of the Pea plant

Rhizobia is a large group of bacteria developing symbiotic relation with their host plants by forming root nodules and fixing atmospheric nitrogen. The bacteria belonging to this group are important from agriculture and environmental perspective. Three strains of rhizobia were collected from Indian Trans-Himalaya region, isolated from root nodules of Pea plant. The characterization was done using 16S rRNA, atpD and recA genes. Based on 16S rRNA gene sequencing, isolated strains showed ≥99.9% sequence similarity to the members within the Rhizobium leguminosarum group. Phylogenetic analysis based on sequences of atpD, recA and 92 core genes indicated that two of the three strains were closely related to the group Rlaguerreae while one strain was closely related to the Rhidalgonense group. Two strains closely related to the group R. laguerreae have 60.8 mol% of DNA G+C content. Based on characterization studies, these two strains represent a novel species, Rhizobium indicum sp.nov. The study provided a detailed taxonomic species description of these novel species.

The nodC and nifH genes play an important role in nodulation and nitrogen fixation respectively. These genes were amplified for all three strains which confirmed their genetic potential for symbiosis.  All three strains have nodC gene sequence similar to the strains of Rhizobium leguminasarum that belong to the symbiovar viciae and isolated from different countries.

Genome studies revealed that the complete genome, chromosome and plasmids of these strains are comparable in size to the other strains of Rhizobium leguminasarum. Researchers also examined the genome sequences of the two strains to identify the compliment of nod genes in these strains. Both strains contained a cluster of 12 nod genes. Phylogenetic analysis revealed that both these strains are closely related to Rhizobium laguerreae followed by Rruizarguesonis and R. leguminosarum.

Morphological analysis revealed that the strains are rod-shaped, gram-negative and motile. Colonies were translucent, cream-white and convex. A total of 20 different cellular fatty acids in different quantities were detected in these three strains. Overall, the fatty acid compositions of the newly isolated strains were identical to the type strains of Rhizobium leguminosarumRhizobium laguerreae and Rhizobium ecuadorense, with differences in their proportions. The strain were positive for oxidase and catalase.

To conclude, both the novel strains are closely related to each other and can be differentiated from other species of the genus Rhizobium by their housekeeping gene sequences, ANI values, genome nucleotide analysis, and phenotypic and chemotaxonomic features.


Description of a novel species isolated from the surface of tomato

-By Kranti Karande

A new bacterial strain designated as TOUT106T was isolated from the tomato surface. Though there were many bacterial species isolated from various fruits and vegetables like sweet potato, banana, tomato, lettuce and cucumber, TOUT106T was found to represent a novel species. This study was aimed at providing a detailed taxonomic description of this novel strain TOUT106T isolated from tomato surface. A novel strain is a micro organism which hasn’t been identified earlier.

The strain was isolated by washing the outer surface of a tomato collected from a local vegetable market in Pune, India. Colonies grown on a trypticase soy agar turned out to be 1-3 mm in diameter, circular, raised with an entire margin, and translucent opacity. The identification was done using MALDI-TOF MS technique. After extracting high quality genomic DNA from the strain, the 16s rRNA sequence was amplified. The similarity search for the 16S rRNA gene sequence of strain TOUT106T was performed against the type strains of prokaryotic species in the EzBioCloud’s database. 

Genome sequencing was performed and from the concatenated sequences of 92 core genes extracted, a possible phylogenetic tree was inferred. The Average Nucleotide Identity (ANI) was determined between strain TOUT106T and closely related strains of the Enterobacteriaceae family. Analysis of chemotaxonomic features (based on fatty acids and cell proteins)was done after harvesting cell biomass from culture grown on TSA at 28°C.  Antibiotic susceptibility was determined using the disc diffusion method. The susceptibility to antibiotics was interpreted based on the Clinical and Laboratory Standards Institute (CLSI) guidelines determined for members of the family Enterobacteriaceae

A search of the 16S rRNA gene sequence of strain TOUT106T showed the highest similarity to Salmonella enterica subsp. arizonae strain NCTC 8297T (98.4 %) . The phylogenetic tree constructed based on the 16S rRNA gene sequence placed the strain TOUT106T within the Salmonella clade. The genomic DNA content of strain TOUT106T was well within the specifications of genus Klebsiella. Comparative analysis of ANI value and dDDH relatedness of Enterobacteriaceae strains suggested that the strain TOUT106T is a novel species.  A comparison of MALDI-TOF MS spectra based dendrogram showed that the strain TOUT106T separated from the type strains of Salmonella and was placed along with Klebsiella variicola DSM 15968T and Raoultella terrigena DSM 2687T, corroborating well with the results of genome-based analysis. Colony morphology, as examined on blood agar medium, were mucoid and translucent.

Based on different methods of identification, scientists reported that the strain TOUT106T is a member of genus Klebsiella. However, it differs from closely related species of the genus Klebsiella in several aspects, such as biochemical features, physiological features, protein profile, and overall genome relatedness indices. Thus, it represents a novel species in the genus Klebsiella, for which the name Klebsiella indica sp. nov. is proposed.

Klebsiella indica (in’ L. fem. adj. indica, of or belonging to India, where the type strain was isolated from the outer wash of a tomato collected from the vegetable market in India). Cells are Gram-negative, straight rods with round ends (0.7-0.9×2-3 μm), and non-motile. Colonies grown on trypticase soy agar are 1-3 mm in diameter, circular, and with translucent opacity. The optimal temperature for growth is 28 °C, and the optimal pH is 7.0. Growth occurs in the absence of NaCl with up to 2% tolerance in trypticase soy broth. It is weakly positive for catalase and negative for oxidase activity. It is susceptible to the majority of antibiotics. 

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