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Genomic Analysis of a Novel Strain of Bacillus Nealsonii, Isolated from Surti Buffalo Rumen

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Genomic Analysis of a Novel Strain of Bacillus Nealsonii, Isolated from Surti Buffalo Rumen Advances in Bioscience and Biotechnology, 2014, 5, 235-245 ABB http://dx.doi.org/10.4236/abb.2014.53030 Published Online February 2014 (http://www.scirp.org/journal/abb/) Genomic analysis of a novel strain of Bacillus nealsonii, isolated from Surti buffalo rumen Neelam M. Nathani1*, Srinivas M. Duggirala2*, Vaibhav D. Bhatt1, Jay KaPatel1, Chaitanya G. Joshi1 1Department of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand, India 2Department of Microbiology, Gujarat Vidyapith, Sadra, India Email: [email protected] Received 9 November 2013; revised 16 January 2014; accepted 28 January 2014 Copyright © 2014 Neelam M. Nathani et al. This is an open access article distributed under the Creative Commons Attribution Li- cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accordance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intel- lectual property Neelam M. Nathani et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian. ABSTRACT posed. Significance and Impact of Study: The strict anaerobic conditions prevailing in the bovine rumen Aim: Whole genome sequencing and functional an- from where AAU1 was isolated may have resulted in notation of Bacillus nealsonii strain AAU1, an amylo- genetic polymorphism influencing its metabolic cha- lytic anaerobic spore forming isolate from ruminal racteristics. contents of buffalo. Methods and Results: Morpho- logically, the strain was observed as slender Gram- KEYWORDS positive rods, occurring in pairs. Optimal growth was observed at 40˚C (range: 30˚C to 45˚C) and pH 6.5 Bacillus nealsonii AAU1; Anaerobic; Phenotypic (range: 5.5 to 7.5) when cultivated in Hungate’s me- Characterization; Genomic Analysis dium supplemented with starch. The microorganism showed extracellular constitutive amylolytic activity, 1. INTRODUCTION proving to be capable of utilizing glucose, maltose, mannose, trehalose, dextrin and starch under an- The rumen harbors a large and diverse range of micro- aerobic conditions. Sequence analysis revealed a GC organisms categorized into Bacteria, Archaea (methano- content of 35.1 mol%. Comparison of housekeeping gens) and Eucarya (protozoa and fungi) [1]. Obligatory gene sequences for RNA polymerase subunit B (rpoB) anaerobes are dominated and supplemented by faculta- and gyrase A (gyrA) identified sequence similarity tive anaerobes including Streptococcus, Staphylococcus, within the Bacillus genus, confirmed by 16S rRNA Bacillus and Lactobacillus species [2]. The complex mi- gene sequence similarity which identified Bacillus ne- crobial ecosystem of the rumen functions as an efficient alsonii DSM 15077 as the closest publically available biological fermentor and provides nutrients essential for relative. Chemotaxonomic analysis provided con- the growth and productivity of the ruminant host in the flicting results with straight-chain saturated C16: form of volatile fatty acids and microbial protein. 0/C16:0 aldehyde, C16:0 DMA, C14:0 and mono- The Bacillus genus, introduced by Cohn in the year unsaturated 16:1w7c and 16:1w9c the major fatty 1872, comprises more than 200 species and is considered acids in contrast to those reported for B. nealsonii to be among the largest bacterial genera with new addi- DSM15077. Further characterization using AN-Bi- tions identified every year. The Bacilli are rod shaped olog and physiological parameters provided genotypic gram positive bacteria, and characterized by spore form- and phenotypic support for taxonomic classification ing ability and aerobic or facultative anaerobic metabol- of isolate AAU1 with published Bacillus species in- ism [3]. Single spores are formed per cell in response to cluding B. licheniformis, B. subtilis, B. circulans and B. environmental stress, such as heat, cold, radiation or de- nealsonii. Conclusion: Based on the data presented, siccation; features which support their existence in ex- isolate is likely to represent a new strain/subspecies, treme habitats include desert sands, hot springs and Arc- for which the identifier B. nealsonii AAU1 is pro- tic soils. Sequencing bacterial genomes provide insight into the genetic basis of phenotypic plasticity and their *Nathani N. M. & Duggirala S. M. equally contribute to the article. ability to tolerate environmental stresses [4]. Bacillus OPEN ACCESS 236 N. M. Nathani et al. / Advances in Bioscience and Biotechnology 5 (2014) 235-245 nealsonii species have previously been isolated from 2.4. Characterization and Substrate potentially harsh environments including a spacecraft Utilization Profile assembly, spores of which were observed to be resistant Carbon utilization by the putative Bacillus isolate was to ultraviolet (UV) light, γ-radiation, hydrogen peroxide and desiccation [5]. Hence, study of these bacteria can investigated biochemically using the AN-Biolog® mi- improve understanding of bacterial responses to envi- croplate assay to determine substrate fermenting poten- ronmental stress and inform upon their role in complex tial as per the manufacturer’s instructions. Subsequently, microbial communities, such as those found within the the OD595 was measured using a microtiter plate reader bovine rumen. after 72 h incubation. Growth, colony characteristics and The Surti buffalo (Bubalus bubalis) is a buffalo breed morphology of the isolate were monitored according to which is popular across Gujarat, India, commonly provi- methods prescribed in Bergey’s Manual. ding milk and draught power. Buffalo are notable for their feed conversion ability from rough grazing, promoting cha- 2.5. FAME Analysis racterization of their ruminal microbiota. We describe Fatty acid methyl ester (FAME) analysis was carried out here the isolation and characterization by sequencing and by gas-liquid chromatography (Sherlock Microbial Iden- biochemical analysis of a putatively new B. nealsonii tification System [MIS]; MIDI, Inc.). Chromatographic strain or isolate with relevance to the future understand- data defining the isolate were analyzed using the Sher- ing and improvement of ruminant health and nutrition. lock software version 6.0B (S/N; 160277) MIDI, with the SMOORE6 method. 2. MATERIALS AND METHOD 2.6. Genome Sequencing and Assembly 2.1. Sampling and Enrichment The whole genome sequence of the putative Bacillus Rumen fluid samples were collected aseptically from a isolate was determined by 454 GS-FLX (Roche) and Ion Surti Buffalo at the Veterinary College, Anand Agricul- Torrent PGM platform sequencing as per the manufac- tural University (AAU), Anand, India. The ruminal fluid turer’s instructions. The results were generated by the GS samples were enriched in Hungate medium containing run browser and the sequencing reads were assembled starch in roll tubes, maintained in strict anaerobic condi- using the GS De Novo Assembler V.2.6 providing con- tions by purging N :CO (80:20) and sealed with butyl 2 2 sensus contigs. rubber and aluminum seals. 2.7. Bioinformatic Analysis: 16S rRNA and 2.2. Media and Culture Conditions Housekeeping Genes Anaerobic culture techniques and incubations were per- Comparison of similarity to published bacterial se- formed as described elsewhere by Hungate and col- quences was confirmed using homology by uploading leagues [6]. Hungate medium [7], used for enrichment, the assembled contigs into the RDP-Ribosomal Database serial dilution and subsequent cultivation of bacteria Project Classifier. For 16S rRNA homology studies, lo- from ruminal fluid, contained (per 1000 ml) K2HPO4 0.5 cal BLAST of the assembled contigs was performed g, KH2PO4 0.2 g, (NH4)2SO4 0.5 g, NaCl 1.0 g, MgSO4 against a 16S rRNA gene sequence database of 7545 0.02 g, CaCl2 0.05 g, NaHCO3 5.0 g, cysteine hydrochlo- sequences downloaded from NCBI. Comparison of genes ride 0.5 g, starch 5.0 g, clarified rumen fluid 1% and agar encoding the housekeeping proteins rpoB (RNA poly- 3% at pH 7.2. The medium was pre gassed with N2:H2 merase open promoter) and gyrase A was undertaken using (80:20); sterilized at 10 psi followed by post gassing with publically available sequences downloaded from NCBI. N2:CO2 (80:20) and sealed with butyl rubber and alumi- Sequences coding for 16S rRNA and gyrA of the genus nium seals. Bacillus were aligned with that of Bacillus nealsonii AAU1, using ClustalW. Subsequently, an evolutionary 2.3. Isolation distance matrix was generated from these nucleotide se- Serial dilutions were prepared in Hungate medium from quences in the dataset using Maximum Composite Like- enriched samples and 0.1 mL from each dilution was lihood method. Phylogenetic analysis was performed streaked on Hungate Agar medium containing starch in using the Neighbor Joining method by MEGA (Molecu- anaerobic bottles. The medium was post gassed with lar Evolutionary Genetics analysis) version 4.0 [8]. N2:CO2 (80:20). All plates were incubated at 40˚C ± 2˚C, after which colony morphology was recorded. Subse- 2.8. Gene Prediction and Annotation quently, three sequential transfers were sub-cultured from Whole genome gene prediction and annotation was per- well isolated colonies after dilution to purify the culture. formed by uploading the assembled contigs onto the Copyright © 2014 SciRes. OPEN ACCESS N. M. Nathani et al. / Advances in Bioscience and Biotechnology 5 (2014) 235-245 237 RAST-Rapid Annotation using Subsystem Technology Table 1. Substrate utilization profile of
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