Substrate preferences, phylogenetic and biochemical properties of proteolytic bacteria present in the digestive tract of Nile tilapia (<i>Oreochromis niloticus</i>)

Tanim Jabid Hossain; Mukta Das; Ferdausi Ali; Sumaiya Islam Chowdhury; Subrina Akter Zedny; Tanim Jabid Hossain; Mukta Das; Ferdausi Ali; Sumaiya Islam Chowdhury; Subrina Akter Zedny

doi:10.3934/microbiol.2021032

AIMS Microbiology

2021, Volume 7, Issue 4: 528-545. doi: 10.3934/microbiol.2021032

Previous Article Next Article

Research article

Substrate preferences, phylogenetic and biochemical properties of proteolytic bacteria present in the digestive tract of Nile tilapia (Oreochromis niloticus)

1.
Department of Biochemistry and Molecular Biology, University of Chittagong, Chattogram 4331, Bangladesh
2.
Biochemistry and Pathogenesis of Microbes Research Group, Chattogram 4331, Bangladesh
3.
Department of Microbiology, University of Chittagong, Chattogram 4331, Bangladesh

Received: 22 October 2021 Accepted: 20 December 2021 Published: 23 December 2021

Vertebrate intestine appears to be an excellent source of proteolytic bacteria for industrial and probiotic use. We therefore aimed at obtaining the gut-associated proteolytic species of Nile tilapia (Oreochromis niloticus). We have isolated twenty six bacterial strains from its intestinal tract, seven of which showed exoprotease activity with the formation of clear halos on skim milk. Their depolymerization ability was further assessed on three distinct proteins including casein, gelatin, and albumin. All the isolates could successfully hydrolyze the three substrates indicating relatively broad specificity of their secreted proteases. Molecular taxonomy and phylogeny of the proteolytic isolates were determined based on their 16S rRNA gene barcoding, which suggested that the seven strains belong to three phyla viz. Firmicutes, Proteobacteria, and Actinobacteria, distributed across the genera Priestia, Citrobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Providencia, and Micrococcus. The isolates were further characterized by a comprehensive study of their morphological, cultural, cellular and biochemical properties which were consistent with the phylogenetic annotations. To reveal their proteolytic capacity alongside substrate preferences, enzyme-production was determined by the diffusion assay. The Pseudomonas, Stenotrophomonas and Micrococcus isolates appeared to be most promising with maximum protease production on casein, gelatin, and albumin media respectively. Our findings present valuable insights into the phylogenetic and biochemical properties of gut-associated proteolytic strains of Nile tilapia.
- Protease producing bacteria,
- gut microbiota,
- Nile tilapia,
- bacterial extracellular protease,
- substrate preference,
- proteolytic activity,
- 16S rRNA gene based phylogeny
Citation: Tanim Jabid Hossain, Mukta Das, Ferdausi Ali, Sumaiya Islam Chowdhury, Subrina Akter Zedny. Substrate preferences, phylogenetic and biochemical properties of proteolytic bacteria present in the digestive tract of Nile tilapia (Oreochromis niloticus)[J]. AIMS Microbiology, 2021, 7(4): 528-545. doi: 10.3934/microbiol.2021032

Related Papers:

Abstract

Vertebrate intestine appears to be an excellent source of proteolytic bacteria for industrial and probiotic use. We therefore aimed at obtaining the gut-associated proteolytic species of Nile tilapia (Oreochromis niloticus). We have isolated twenty six bacterial strains from its intestinal tract, seven of which showed exoprotease activity with the formation of clear halos on skim milk. Their depolymerization ability was further assessed on three distinct proteins including casein, gelatin, and albumin. All the isolates could successfully hydrolyze the three substrates indicating relatively broad specificity of their secreted proteases. Molecular taxonomy and phylogeny of the proteolytic isolates were determined based on their 16S rRNA gene barcoding, which suggested that the seven strains belong to three phyla viz. Firmicutes, Proteobacteria, and Actinobacteria, distributed across the genera Priestia, Citrobacter, Pseudomonas, Stenotrophomonas, Burkholderia, Providencia, and Micrococcus. The isolates were further characterized by a comprehensive study of their morphological, cultural, cellular and biochemical properties which were consistent with the phylogenetic annotations. To reveal their proteolytic capacity alongside substrate preferences, enzyme-production was determined by the diffusion assay. The Pseudomonas, Stenotrophomonas and Micrococcus isolates appeared to be most promising with maximum protease production on casein, gelatin, and albumin media respectively. Our findings present valuable insights into the phylogenetic and biochemical properties of gut-associated proteolytic strains of Nile tilapia.

Acknowledgments

We are grateful to Prof. Dr. Md. Monirul Islam, Dept. of Biochemistry and Molecular Biology, University of Chittagong (BMB, CU) for his kind support with laboratory equipment. We thank members of the Biochemistry and Pathogenesis of Microbes–BPM Research Group, BMB, CU for their various help in this project.

Author contributions

TJH conceived and designed the study; MD, FA, TJH and SIC performed experiments; SIC contributed to sequence study and TJH performed the phylogenetic analysis; TJH and FA interpreted the data; TJH wrote and prepared the manuscript; SAZ assisted in writing introduction; SAZ and FA helped in information collection; all authors approved the final manuscript.

Funding

This research was supported by University of Chittagong via its Planning and Development Department to TJH.

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

This article does not contain any experiments performed on human participants or animals by any of the authors.

References

[1]	Jakubke HD, Kuhl P, Könnecke A (1985) Basic Principles of protease-catalyzed peptide bond formation. Angewandte Chemie International Edition in English 24: 85-93. doi: 10.1002/anie.198500851
[2]	Gupta R, Beg Q, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59: 15-32. doi: 10.1007/s00253-002-0975-y
[3]	Waschkowitz T, Rockstroh S, Daniel R (2009) Isolation and characterization of metalloproteases with a novel domain structure by construction and screening of metagenomic libraries. Appl Environ Microbiol 75: 2506-2516. doi: 10.1128/AEM.02136-08
[4]	Razzaq A, Shamsi S, Ali A, et al. (2019) Microbial proteases applications. Front Bioeng Biotechnol 7: 110. doi: 10.3389/fbioe.2019.00110
[5]	Zhu D, Wu Q, Hua L (2019) Industrial enzymes. Comprehensive Biotechnology Oxford: Pergamon, 1-13.
[6]	Graves PR, Haystead TAJ (2002) Molecular biologist's guide to proteomics. Microbiol Mol Biol Rev 66: 39-63. doi: 10.1128/MMBR.66.1.39-63.2002
[7]	Białkowska AM, Morawski K, Florczak T (2017) Extremophilic proteases as novel and efficient tools in short peptide synthesis. J Ind Microbiol Biotechnol 44: 1325-1342. doi: 10.1007/s10295-017-1961-9
[8]	Yang H, Li YC, Zhao MZ, et al. (2019) Precision de novo peptide sequencing using mirror proteases of Ac-LysargiNase and trypsin for large-scale proteomics. Mol Cell Proteomics 18: 773-785. doi: 10.1074/mcp.TIR118.000918
[9]	Theron LW, Divol B (2014) Microbial aspartic proteases: current and potential applications in industry. Appl Microbiol Biotechnol 98: 8853-8868. doi: 10.1007/s00253-014-6035-6
[10]	Eun HM (1996) 6-DNA Polymerases. Enzymology Primer for Recombinant DNA Technology San Diego: Academic Press, 345-489. doi: 10.1016/B978-012243740-3/50009-0
[11]	Olajuyigbe FM, Falade AM (2014) Purification and partial characterization of serine alkaline metalloprotease from Bacillus brevis MWB-01. Bioresour Bioprocess 1: 8. doi: 10.1186/s40643-014-0008-6
[12]	Cui H, Yang M, Wang L, et al. (2015) Identification of a new marine bacterial strain SD8 and optimization of its culture conditions for producing alkaline protease. PLOS One 10: e0146067. doi: 10.1371/journal.pone.0146067
[13]	Martínez-Medina GA, Barragán AP, Ruiz HA, et al. (2019) Fungal Proteases and Production of Bioactive Peptides for the Food Industry. Enzymes in Food Biotechnology Cambridge: Academic Press, 221-246. doi: 10.1016/B978-0-12-813280-7.00014-1
[14]	Tacon AGJ (2020) Trends in global aquaculture and aquafeed production: 2000–2017. Rev Fish Sci Aquacult 28: 43-56. doi: 10.1080/23308249.2019.1649634
[15]	Hossain TJ, Chowdhury SI, Mozumder HA, et al. (2020) Hydrolytic exoenzymes produced by bacteria isolated and identified from the gastrointestinal tract of Bombay duck. Front Microbiol 11. doi: 10.3389/fmicb.2020.02097
[16]	Selim KM, Reda RM (2015) Improvement of immunity and disease resistance in the Nile tilapia, Oreochromis niloticus, by dietary supplementation with Bacillus amyloliquefaciens. Fish Shellfish Immunol 44: 496-503. doi: 10.1016/j.fsi.2015.03.004
[17]	Su H, Xiao Z, Yu K, et al. (2020) Diversity of cultivable protease-producing bacteria and their extracellular proteases associated to scleractinian corals. PeerJ 8: e9055. doi: 10.7717/peerj.9055
[18]	Amin M (2018) Marine protease-producing bacterium and its potential use as an abalone probiont. Aquacult Rep 12: 30-35. doi: 10.1016/j.aqrep.2018.09.004
[19]	Maas RM, Deng Y, Dersjant-Li Y, et al. (2021) Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia. Sci Rep 11: 8221. doi: 10.1038/s41598-021-87408-3
[20]	Anshary H, Kurniawan RA, Sriwulan S, et al. (2014) Isolation and molecular identification of the etiological agents of streptococcosis in Nile tilapia (Oreochromis niloticus) cultured in net cages in Lake Sentani, Papua, Indonesia. SpringerPlus 3: 627. doi: 10.1186/2193-1801-3-627
[21]	Champneys T, Castaldo G, Consuegra S, et al.Density-dependent changes in neophobia and stress-coping styles in the world's oldest farmed fish. R Soc Open Sci 5: 181473. doi: 10.1098/rsos.181473
[22]	Njiru M, Okeyo-Owuor J, Muchiri M, et al. (2004) Shifts in the food of Nile tilapia, Oreochromis niloticus (L.) in Lake Victoria, Kenya. Afr J Ecol 42: 163-170. doi: 10.1111/j.1365-2028.2004.00503.x
[23]	Moyle PB, Cech JJ (2004) Fishes: An Introduction to Ichthyology New Jersey: Pearson Prentice Hall, 559.
[24]	BPM BPM Research Group, Bacteriological Growth Media: Composition, Preparation and Preservation of Nutritional Media for Culturing Bacteria, 2020 (2020) .Available from: https://sites.google.com/view/bpm-research-group/research/media-composition.
[25]	Hossain TJ, Alam MK, Sikdar D (2011) Chemical and microbiological quality assessment of raw and processed liquid market milks of Bangladesh. Cont J Food Sci Technol 5: 6-17.
[26]	Carter GR (1990) Isolation and identification of bacteria from clinical specimens. Diagnostic procedure in veterinary bacteriology and mycology Elsevier, 19-39. doi: 10.1016/B978-0-12-161775-2.50008-6
[27]	Zhang Z, Schwartz S, Wagner L, et al. (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7: 203-214. doi: 10.1089/10665270050081478
[28]	Wang Q, Garrity GM, Tiedje JM, et al. (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261-5267. doi: 10.1128/AEM.00062-07
[29]	Pruesse E, Peplies J, Glöckner FO (2012) SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28: 1823-1829. doi: 10.1093/bioinformatics/bts252
[30]	Hossain TJ, Manabe S, Ito Y, et al. (2018) Enrichment and characterization of a bacterial mixture capable of utilizing C-mannosyl tryptophan as a carbon source. Glycoconjugate J 35: 165-176. doi: 10.1007/s10719-017-9807-2
[31]	Schoch CL, Ciufo S, Domrachev M, et al. (2020) NCBI Taxonomy: a comprehensive update on curation, resources and tools. Database (Oxford) 2020: baaa062. doi: 10.1093/database/baaa062
[32]	Ali Ferdausi, Das Sharup, Hossain Tanim Jabid, et al. (2021) Production optimization, stability, and oil emulsifying potential of biosurfactants from selected bacteria isolated from oil contaminated sites. R Soc Open Sci 8.
[33]	Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792-1797. doi: 10.1093/nar/gkh340
[34]	Kumar S, Stecher G, Li M, et al. (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547-1549. doi: 10.1093/molbev/msy096
[35]	(2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67: 1613-1617.
[36]	Hasegawa M, Kishino H, Saitou N (1991) On the maximum likelihood method in molecular phylogenetics. J Mol Evol 32: 443-445. doi: 10.1007/BF02101285
[37]	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.
[38]	Garrity GM, Bell JA, Lilburn TG (2004) Taxonomic outline of the prokaryotes. Bergey's manual of systematic bacteriology New York: Springer-Verlag.
[39]	Reda RM, Selim KM, El-Sayed HM, et al. (2018) In vitro selection and identification of potential probiotics isolated from the gastrointestinal tract of nile tilapia, Oreochromis niloticus. Probiotics Antimicrob Proteins 10: 692-703. doi: 10.1007/s12602-017-9314-6
[40]	Bairagi A, Ghosh KS, Sen SK, et al. (2002) Enzyme producing bacterial flora isolated from fish digestive tracts. Aquacult Int 10: 109-121. doi: 10.1023/A:1021355406412
[41]	Kar N, Ghosh K (2008) Enzyme producing bacteria in the gastrointestinal tracts of Labeo rohita (Hamilton) and Channa punctatus (Bloch). Turkish J Fish Aquat Sci 8: 115-120.
[42]	Molinari L, Scoaris D, Pedroso R, et al. (2003) Bacterial microflora in the gastrointestinal tract of Nile tilapia, Oreochromis niloticus, cultured in a semi-intensive system. Acta Sci Biol Sci 25: 267-271.
[43]	Saha S, Roy RN, Sen SK, et al. (2006) Characterization of cellulase-producing bacteria from the digestive tract of tilapia, Oreochromis mossambica (Peters) and grass carp, Ctenopharyngodon idella (Valenciennes). Aquacult Res 37: 380-388. doi: 10.1111/j.1365-2109.2006.01442.x
[44]	Wu F, Chen B, Liu S, et al. (2020) Effects of woody forages on biodiversity and bioactivity of aerobic culturable gut bacteria of tilapia (Oreochromis niloticus). PLOS One 15: e0235560. doi: 10.1371/journal.pone.0235560
[45]	Haygood AM, Jha R (2018) Strategies to modulate the intestinal microbiota of Tilapia (Oreochromis sp.) in aquaculture: a review. Rev Aquacult 10: 320-333. doi: 10.1111/raq.12162
[46]	Afrilasari W, Widanarni, Meryandini A (2016) Effect of probiotic Bacillus megaterium PTB 1.4 on the population of intestinal microflora, digestive enzyme activity and the growth of catfish (Clarias sp.). HAYATI J Biosci 23: 168-172. doi: 10.1016/j.hjb.2016.12.005
[47]	Zorriehzahra MJ, Delshad ST, Adel M, et al. (2016) Probiotics as beneficial microbes in aquaculture: an update on their multiple modes of action: a review. Null 36: 228-241.
[48]	Yang C, Jiang M, Lu X, et al. (2021) Effects of dietary protein level on the gut microbiome and nutrient metabolism in tilapia (Oreochromis niloticus). Animals 11: 1024. doi: 10.3390/ani11041024
[49]	Zaky MMM, Ibrahim ME (2017) Screening of bacterial and fungal biota associated with Oreochromis niloticus in Lake Manzala and its impact on human health. Health 9: 697-714. doi: 10.4236/health.2017.94050
[50]	Boari CA, Pereira GI, Valeriano C, et al. (2008) Bacterial ecology of tilapia fresh fillets and some factors that can influence their microbial quality. Food Sci Technol 28: 863-867. doi: 10.1590/S0101-20612008000400015
[51]	Biedendieck R, Knuuti T, Moore SJ, et al. (2021) The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology. Appl Microbiol Biotechnol 105: 5719-5737. doi: 10.1007/s00253-021-11424-6
[52]	Nicodème M, Grill JP, Humbert G, et al. (2005) Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions. J Appl Microbiol 99: 641-648. doi: 10.1111/j.1365-2672.2005.02634.x
[53]	Asker MMS, Mahmoud MG, El Shebwy K, et al. (2013) Purification and characterization of two thermostable protease fractions from Bacillus megaterium. J Genet Eng Biotechnol 11: 103-109. doi: 10.1016/j.jgeb.2013.08.001
[54]	Ray AK, Roy T, Mondal S, et al. (2010) Identification of gut-associated amylase, cellulase and protease-producing bacteria in three species of Indian major carps. Aquacult Res 41: 1462-1469.
[55]	Lee MA, Liu Y (2000) Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomallei. FEMS Microbiol Lett 192: 67-72. doi: 10.1111/j.1574-6968.2000.tb09360.x
[56]	Miyaji T, Otta Y, Shibata T, et al. (2005) Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S-1. Lett Appl Microbiol 41: 253-257. doi: 10.1111/j.1472-765X.2005.01750.x
[57]	Bhowmik T, Marth EH (1988) Protease and peptidase activity of Micrococcus species. J Dairy Sci 71: 2358-2365. doi: 10.3168/jds.S0022-0302(88)79819-7
[58]	Rodarte MP, Dias DR, Vilela DM, et al. (2011) Proteolytic activities of bacteria, yeasts and filamentous fungi isolated from coffee fruit (Coffea arabica L.). Acta Sci Agron 33: 457-464.
[59]	Zeng A, Tan K, Gong P, et al. (2020) Correlation of microbiota in the gut of fish species and water. 3 Biotech 10: 472. doi: 10.1007/s13205-020-02461-5
[60]	Kim PS, Shin NR, Lee JB, et al. (2021) Host habitat is the major determinant of the gut microbiome of fish. Microbiome 9: 166. doi: 10.1186/s40168-021-01113-x
[61]	Liu H, Guo X, Gooneratne R, et al. (2016) The gut microbiome and degradation enzyme activity of wild freshwater fishes influenced by their trophic levels. Sci Rep 6: 24340. doi: 10.1038/srep24340
[62]	Burtseva O, Kublanovskaya A, Fedorenko T, et al. (2021) Gut microbiome of the White Sea fish revealed by 16S rRNA metabarcoding. Aquaculture 533: 736175. doi: 10.1016/j.aquaculture.2020.736175
[63]	Egerton S, Culloty S, Whooley J, et al. (2018) The gut microbiota of marine fish. Front Microbiol 9. doi: 10.3389/fmicb.2018.00873
[64]	Bereded N, Curto M, Domig K, et al. (2020) Metabarcoding analyses of gut microbiota of Nile tilapia (Oreochromis niloticus) from Lake Awassa and Lake Chamo, Ethiopia. Microorganisms 8: 1040. doi: 10.3390/microorganisms8071040
[65]	Hassaan MS, Mohammady EY, Soaudy MR, et al. (2021) Synergistic effects of Bacillus pumilus and exogenous protease on Nile tilapia (Oreochromis niloticus) growth, gut microbes, immune response and gene expression fed plant protein diet. Anim Feed Sci Technol 275: 114892. doi: 10.1016/j.anifeedsci.2021.114892
[66]	Wang M, Liu G, Lu M, et al. (2017) Effect of Bacillus cereus as a water or feed additive on the gut microbiota and immunological parameters of Nile tilapia. Aquacult Res 48: 3163-3173. doi: 10.1111/are.13146
[67]	Xia Y, Wang M, Gao F, et al. (2020) Effects of dietary probiotic supplementation on the growth, gut health and disease resistance of juvenile Nile tilapia (Oreochromis niloticus). Anim Nutr 6: 69-79. doi: 10.1016/j.aninu.2019.07.002
[68]	Giatsis C, Sipkema D, Smidt H, et al. (2015) The impact of rearing environment on the development of gut microbiota in tilapia larvae. Sci Rep 5: 18206. doi: 10.1038/srep18206
[69]	Bereded NK, Abebe GB, Fanta SW, et al. (2021) The Impact of sampling season and catching site (wild and aquaculture) on gut microbiota composition and diversity of Nile tilapia (Oreochromis niloticus). Biology (Basel) 10: 180.
[70]	Jaouadi NZ, Rekik H, Badis A, et al. (2013) Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities. PLOS One 8: e76722. doi: 10.1371/journal.pone.0076722
[71]	Li HJ, Tang BL, Shao X, et al. (2016) Characterization of a New S8 serine protease from marine sedimentary photobacterium sp. A5–7 and the function of its protease-associated domain. Front Microbiol 7: 2016.
[72]	Saggu SK, Jha G, Mishra PC (2019) Enzymatic degradation of biofilm by metalloprotease from Microbacterium sp. SKS10. Front Bioeng Biotechnol 7: 192. doi: 10.3389/fbioe.2019.00192
[73]	Zhou C, Qin H, Chen X, et al. (2018) A novel alkaline protease from alkaliphilic Idiomarina sp. C9-1 with potential application for eco-friendly enzymatic dehairing in the leather industry. Sci Rep 8: 16467. doi: 10.1038/s41598-018-34416-5
[74]	Yildirim V, Baltaci MO, Ozgencli I, et al. (2017) Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents. J Enzyme Inhib Med Chem 32: 468-477. doi: 10.1080/14756366.2016.1261131
[75]	Chellappan S, Jasmin C, Basheer SM, et al. (2011) Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10. J Ind Microbiol Biotechnol 38: 743-752. doi: 10.1007/s10295-010-0914-3
[76]	Niyonzima FN, More SS (2015) Purification and characterization of detergent-compatible protease from Aspergillus terreus gr. 3 Biotech 5: 61-70. doi: 10.1007/s13205-014-0200-6

microbiol-07-04-032-S001.pdf

Reader Comments

Your name:*

Email:*
© 2021 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)