16S rRNA gene sequencing and MALDI-TOF mass spectrometry based comparative assessment and bioprospection of psychrotolerant bacteria isolated from high altitudes under mountain ecosystem

Abstract

The Himalayan Mountains are placed among the globally recognized biodiversity hot spots. While the Indian Himalayan Region (IHR) has been subjected to extensive studies on plant and animal biodiversity, microbial diversity is now being studied for its bioprospection. The present paper deals with the evaluation of bacterial diversity in high-altitude soil samples from IHR following polyphasic approach including comparison between the MALDI-TOF mass spectrometry and 16S rRNA gene sequencing for species-level identification. Initially, a culture collection of large number of bacterial isolates was established in the laboratory. Performing morphological and biochemical screenings, sixty-one representative isolates were selected for mass spectrometry and gene sequencing. Both the methods emerged with bacterial identification showing maximum number of Bacillus followed by Pseudomonas species. The other frequently isolated strains belonged to the genera Alcaligenes, Carnobacterium, Lysinibacillus, Microbacterium, Paenarthrobacter, Rhodococcus, Serratia and Stenotrophomonas. Although the MALDI-TOF technique appeared to be advantageous as less time-consuming in comparison with 16S rRNA-based method, the discrepancies at species level indicated the limited database of MALDI Biotyper and species complexity in the genera. The remarkable characteristics of the bacterial isolates were their tolerance to wide range of pH and temperature. Their potential to produce industrially valuable enzymes indicated their importance in bioprospection. Accessioning of these bacterial isolates in microbial culture collections is a cautious effort for their availability to conduct advanced research on these cold-adapted bacteria in future.

Habitat Elevation Shapes Microbial Community Composition and Alter the Metabolic Functions in Wild Sable (Martes zibellina) Guts

“…However, due to the robustness and resilience of the gut microbial community, wild sables can still survive under such conditions. Similar conclusions were reached by previous studies on gut microbiota of black-necked cranes ( Grus nigricollis ) [ 64 ] and Tibet wild asses ( Equus kiang ) [ 70 ] on the Qinghai-Tibet plateau. Taken together, this study shows that wild sables are adaptable to changing conditions and resistant to harsh environments, and that this adaptability is mediated by gut bacterial composition adjustment and metabolic functions enhancement.…”

“…This finding is consistent with a previous study on black-necked cranes gut microbiota [ 63 ]. Among genera with the ability for producing lipase, a focus had been on enzyme produced by members of the genus Pseudomonas [ 64 ], and the most dominant species of Pseudomonas in sable gut was Pseudomonas fragi , which is a psychrophilic microorganism and able to produce lipase with high catalytic activity at low temperatures [ 62 ]. It is reasonable to conclude that the lower temperature at higher altitudes induced sables demand more low-temperature adaptable lipases to decompose and digest fat, which in turn requires more Pseudomonas fragi producing cold active lipases.…”

“…For example, the ability for energy metabolism and fermentation of polysaccharides in house mice gut microbiota was enhanced with increased elevation [ 54 ], and gut microbiota of rhesus macaques living at higher altitudes had higher expression of genes related to energy metabolism and lipid metabolism [ 34 ]. A possible interpretation is that altitude usually affects other environmental factors such as temperature, oxygen levels, ultraviolet radiation, wind, etc., and it also affects biological factors such as vegetation type, canopy density and concealment level, which in turn affected the physiological characteristics of wild animals [ 64 ]. In our study area, the temperature, which was the most relevant and crucial variable related to altitude, significantly decreased from bottom to top, and the maximum temperature difference across this area could reach 15 °C.…”

Development of an inexpensive matrix-assisted laser desorption—time of flight mass spectrometry method for the identification of endophytes and rhizobacteria cultured from the microbiome associated with maize

“…For this study, the percentage of bacteria identified to the probable species level (32%) with the Biotyper system was lower than previous reports (73%) for soil bacteria (Strejcek et al, 2018) but in the range reported for a library generated from the rhizosphere of horseradish (Uhlik et al, 2011) and bacteria isolated from seawater (Timperio et al, 2017). We also observed discrepancies between species identifications for the two approaches, as reported previously and confirmed recently (Pandey et al, 2019). This could reflect the paucity of environmental isolates in the reference library we queried.…”

Microbial Ecology from the Himalayan Cryosphere Perspective

“…The microbial communities were found to be mainly associated with the biogeochemical cycle of carbon and nitrogen [53]. In a recent study, the culture-dependent bacterial diversity (high abundance of Firmicutes and Proteobacteria) of the glacial sites of Himalaya has been reported, which showed the importance of Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) mass spectrometry along with nucleic acid based methods, in the identification of cold-tolerant bacterial strains [54].…”