Skip to main content
SpringerLink
Log in

Polyphasic characterization of and genomic insights into a haloalkali-tolerant Saccharibacillus alkalitolerans sp. nov., that produces three cellulase isozymes and several antimicrobial compounds

  • Original Paper
  • Published:
Antonie van Leeuwenhoek Aims and scope Submit manuscript

Abstract

A cellulase producing novel bacterial strain VR-M41T was isolated from an open-air vegetable and fruit market. Cells are found to be rod-shaped, endospore forming, positive for Gram’s stain and negative for catalase, oxidase and urease. Strain VR-M41T was halotolerant (upto 8.0% NaCl, w/v), motile and facultative anaerobe. It grew at wide range of pH (6.0–10.0) and temperatures (20–40 °C). Strain VR-M41T produced three isozymes of Carboxymethylcellulase. The 16S rRNA gene sequence of strain VR-M41T was 97.3% similar to both Saccharibacillus kuerlensis DSM 22868T and Saccharibacillus sacchari DSM 19268T, and less than 96.4% with the rest of the valid species of the genus Saccharibacillus. Whole-genome ANI, dDDH and genome phylogenetic tree analysis revealed that strain VR-M41T significantly differed from Saccharibacillus kuerlensis DSM 22868T and Saccharibacillus sacchari DSM 19268T (ANI 79.6–79.7% and dDDH 23.1%). The strain comprised of MK-7 and anteiso-C 15:0 (42.2%) as predominant isoprenoid quinone and fatty acid respectively. Major polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The draft genome of strain VR-M41T consisted of 5,386,426 base pairs with 5103 annotated genes, out of which 2147 corresponded to hypothetical proteins and 2956 with functional assignments. Pan-genome analysis revealed the presence of 2998 core genes, 828 accessory genes, and 1131 unique genes of Saccharibacillus. Strain VR-M41T produced antimicrobials against Staphylococcus aureus, Streptococcus pneumoniae, Micrococcus luteus and Shigella flexneri. Significant phenotypic and genotypic differentiating characteristics from closely related species, indicated that strain VR-M41T is a novel species of the genus Saccharibacillus, for which the name Saccharibacillus alkalitolerans sp. nov., is proposed. The type strain is VR-M41T (= KCTC 43183T=NBRC 114337T).

Graphic Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

All the data included in this study are available with the corresponding author.

Abbreviations

CMCase:

Carboxymethylcellulase

ANI:

Average Nucleotide Identity

dDDH:

Digital DNA-DNA hybridization

BPGA:

The Bacterial Pan Genome Analysis

NJ:

Neighbor-joining

References

  • http://ggdc.dsmz.de/distcalc2.php (Accessed on 05–07–2020)

  • http://www.ezbiocloud.net/sw/oat (Accessed on 05–02–2020)

  • https://lpsn.dsmz.de/search?word=saccharibacillus (Accessed on 06–01–2021)

  • Ayitso AS, Onyango DM (2016) Isolation and identification by morphological and biochemical methods of antibiotic producing microorganisms from the gut of Macrotermes michaelseni in Maseno, Kenya. J Appl Biol Biotechnol 4:27–33

    CAS  Google Scholar 

  • Bashiri G, Johnson JM, Evans GL, Bulloch EMM, Goldstone DC, Jirgis ENM, Kleinboelting S, Castell A, Ramsay RJ, Manos-Turvey A, Payne RJ et al (2015) Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex. Acta Cryst 71:2297–2308

    CAS  Google Scholar 

  • Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, Medema MH, Weber T (2019) antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res 47:W81–W87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Card GL (1973) Metabolism of phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin of Bacillus stearothermophilus. J Bacteriol 114:1125–1137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • CLSI, C (2012) Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-CLSI document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500

  • Costa MSda, Albuquerque L, Nobre MF, Wait R (2011) The extraction and identification of respiratory lipoquinones of prokaryotes and their use in taxonomy. In: Rainey F, Oren A (eds) Taxonomy of Prokaryotes. Academic Press, Methods in Microbiology, pp 197–206

    Chapter  Google Scholar 

  • Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, Néron B, Rocha EPC, Vergnaud G, Gautheret D, Pourcel C (2018) CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res 46:W246–W251

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Funke G, Funke-Kissling P (2004) Use of the BD PHOENIX automated identification and susceptibility testing positive blood cultures in a microbiology system for direct of Gram-negative rods from three-phase trial. J Clin Microbiol 42:1466–1470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gufe C, Canaan-Hodobo T, Mbonjani B, Majonga O, Marumure J, Musari S, Jongi G, Makaya PV, Machakwa J (2019) Antimicrobial profiling of bacteria isolated from fish sold at informal market in Mufakose, Zimbabwe. Int J Microbiol 2019:1–7

    Article  Google Scholar 

  • Jaillard M, van Belkum A, Cady KC, Creely D, Shortridge D, Blanc B, Barbu EM, Dunne WM Jr, Zambardi G, Enright M et al (2017) Correlation between phenotypic antibiotic susceptibility and the resistome in Pseudomonas aeruginosa. Int J Antimicrob Agents 50:210–218

    Article  CAS  PubMed  Google Scholar 

  • Kruger NJ (2009) The Bradford method for protein quantitation. In: Walker JM (ed) The Protein Protocols Handbook. Humana Press, NJ, pp 17–24

    Chapter  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp 115–175

    Google Scholar 

  • Lee I, Ouk Kim Y, Park SC, Chun J (2016) OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66:1100–1103

    Article  CAS  PubMed  Google Scholar 

  • Lozano C, Azcona-Gutierrez JM, Van Bambeke F, Saenz Y (2018) Great phenotypic and genetic variation among successive chronic Pseudomonas aeruginosa from a cystic fibrosis patient. PLoS ONE 13:1–15

    Google Scholar 

  • Lugani Y, Singla R, Sooch BS (2015) Optimization of cellulase production from mewly isolated Bacillus sp. Y3. J Bioprocess Biotech 5:1–6

    Article  Google Scholar 

  • MacFaddin JF (2000) Biochemical tests for identification of medical bacteria, 3rd edn. Lippincott Williams and Wilkins, Philadelphia

    Google Scholar 

  • Marmur JA (1961) Procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Bio 3:208–218

    Article  CAS  Google Scholar 

  • McDevitt S (2009) Methyl Red and Voges-Proskauer test protocols. Am Soc Microbiol pp 1–9

  • Meier-Kolthoff JP, Auch AF, Klenk HP, Goker M (2014) Highly parallelized inference of large genome-based phylogenies. Concurr Comp Pract E 26:1715–1729

    Article  Google Scholar 

  • Merino E, Jensen RA, Yanofsky C (2008) Evolution of bacterial trp operons and their regulation. Curr Opin Microbiol 11:78–86

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miller GL, Blum R, Glennon WE, Burton AL (1960) Measurement of carboxymethylcellulase activity. Anal Biochem 1:127–132

    Article  CAS  Google Scholar 

  • Navraj S, Tariq M, Aruna K (2016) Optimization of inulinase production by Stenotrophomonas maltophila D457 isolated from rhizosphere soil of Musa acuminata using garlic extract. Int J Res Stu Microbiol Biotechnol 2:1–14

    Google Scholar 

  • Nei M, Kumar S (2000) Molecular evolution and phylogenetics. Oxford University Press, New York

    Google Scholar 

  • Oumer OJ, Abate D (2018) Screening and molecular identification of pectinase producing microbes from coffee pulp. Biomed Res Int 2018:1–7

    Google Scholar 

  • Patil VS, Salunkhe RC, Patil RH, Husseneder C, Shouche YS, Ramana VV (2015) Enterobacillus tribolii gen. nov., sp. nov., a novel member of the family Enterobacteriaceae, isolated from the gut of a red flour beetle Tribolium castaneum. Antonie Van Leeuwenhoek 107:1207–1216

    Article  CAS  PubMed  Google Scholar 

  • Pawlowski AC, Wang WL, Koteva K, Barton HA, McArthur AG, Wright GD (2016) A diverse intrinsic antibiotic resistome from a cave bacterium. Nat Commun 7:1–10

    Article  Google Scholar 

  • Pinjari AB, Kotari V (2018) Characterization of extracellular amylase from Bacillus sp. strain RU1. J Appl Biol Biotechnol 6:29–34

    CAS  Google Scholar 

  • Ramana VV, Sasikala C, Takaichi S, Ramana CV (2010) Roseomonas aestuarii sp. nov., a bacteriochlorophyll-a containing alpha proteobacterium isolated from an estuarine habitat of India. Syst Appl Microbiol 33:198–203

    Article  CAS  PubMed  Google Scholar 

  • Reller LB, Weinstein M, Jorgensen JH, Ferraro MJ (2009) Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis 49:1749–1755

    Article  Google Scholar 

  • Rivas R, Garcia-Fraile P, Zurdo-Pineiro JL, Mateos PF, Martinez-Molina E, Bedmar EJ, Sanchez-Raya J, Velazquez E (2008) Saccharibacillus sacchari gen. nov., sp. nov., isolated from sugar cane. Int J Syst Evol Microbiol 58:1850–1854

    Article  CAS  PubMed  Google Scholar 

  • Rzhetsky A, Nei M (1992) A simple method for estimating and testing minimum-evolution trees. Mol Biol Evol 9:945

    CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Sambrook J, Fritschi EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Silva OC, Kosuge T (1991) Molecular characterization and expression analysis of the anthranilate synthase gene of Pseudomonas syringae subsp. savastanoi. J Bacteriol 173:463–471

    Article  Google Scholar 

  • Sherlock Microbial Identification System. MIDI. Version 6.2. MIS Operating Manual. September 2012. www.midi-inc.com

  • Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526

    CAS  PubMed  Google Scholar 

  • Tindall BJ (1990a) A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13:128–130

    Article  CAS  Google Scholar 

  • Tindall BJ (1990b) Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66:199–202

    Article  CAS  Google Scholar 

  • Tomme P, Warren RAJ, Gilkes NR (1995) Cellulose hydrolysis by bacteria and fungi. In: Poole RK (ed) Advances in microbial physiology. Academic Press, USA

    Google Scholar 

  • Tsegaye B, Balomajumder C, Roy P (2019) Microbial delignification and hydrolysis of lignocellulosic biomass to enhance biofuel production: an overview and future prospect. Bull Natl Res Cent 43:51

    Article  Google Scholar 

  • Verma A, Pal Y, Kumar P, Krishnamurthi S (2020) Halocatena pleomorpha gen. nov. sp. nov., an extremely halophilic archaeon of family Halobacteriaceae isolated from saltpan soil. Int J Syst Evol Microbiol 70:3693–3700

    Article  CAS  PubMed  Google Scholar 

  • Yang SY, Liu H, Liu R, Zhang KY, Lai R (2009) Saccharibacillus kuerlensis sp. nov., isolated from a desert soil. Int J Syst Evol Microbiol 59:953–957

    Article  CAS  PubMed  Google Scholar 

  • Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67:1613–1617

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgement

We thank the Council of Scientific and Industrial Research (CSIR), the Director of IMTECH, Chandigarh and Microbial Type Culture Collection and Gene Bank (MTCC) for financial assistance and infrastructural facilities. Priyam Mehrotra thanks University Grants Commission (UGC) for JRF fellowship, Dev Kant Sindhu thanks CSIR for SRF fellowship. Dr. Avinash Sharma, Mr. Kunal Jani and Dr. Yogesh S Shouche are acknowledged for helping in whole genome sequence analysis.

Funding

The current work was supported by funds received from the CSIR-IMTECH institutional project OLP-805. The CSIR-IMTECH communication number is 009/2020.

Author information

Authors and Affiliations

  1. Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, 160036, India

    Himanshu Darji, Neha Verma, Yogita Lugani, Priyam Mehrotra, Dev Kant Sindhu & Venkata Ramana Vemuluri

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Neha Verma, Priyam Mehrotra & Venkata Ramana Vemuluri

Authors
  1. Himanshu Darji
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Neha Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yogita Lugani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Priyam Mehrotra
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dev Kant Sindhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Venkata Ramana Vemuluri
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

VRV conceptualized and designed the study. Experiments performed by HD, NV, YL, PM, and DS. Data analysed by VRV and HD. VRV wrote the manuscript and assisted by YL.

Corresponding author

Correspondence to Venkata Ramana Vemuluri.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Himanshu Darji, Neha Verma, Yogita Lugani, Priyam Mehrotra and Dev Kant Sindhu have contributed equally to the study

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 641 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Darji, H., Verma, N., Lugani, Y. et al. Polyphasic characterization of and genomic insights into a haloalkali-tolerant Saccharibacillus alkalitolerans sp. nov., that produces three cellulase isozymes and several antimicrobial compounds. Antonie van Leeuwenhoek 114, 1043–1057 (2021). https://doi.org/10.1007/s10482-021-01575-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10482-021-01575-x

Keywords

Search

Navigation