Epilithic Biofilms in Lake Baikal: Screening and Diversity of PKS and NRPS Genes in the Genomes of Heterotrophic Bacteria

Taxonomy		Number of the strains analyzed	Number of the strains with positive PCR signal
Phylum	Genus	Number of the strains analyzed	PKS	NRPS
Firmicutes	Bacillus	42	11	33
	Paenibacillus	4	4	4
	Virgibacillus	1	0	0
	Staphylococcus	2	0	1
Proteobacteria	Pseudomonas	45	9	26
	Aeromonas	29	4	2
	Serratia	3	2	1
	Rhizobium	1	1	0
	Brevundimonas	1	0	0
	Massilia	1	0	0
	Achromobacter	3	0	0
	Stenotrophomonas	3	0	0
	Devosia	1	1	0
	Hydrogenophaga	1	0	0
	Yersinia	1	1	0
	Sphingomonas	1	0	0
	Iodobacter	1	1	0
	Roseomonas	1	0	0
Actinobacteria	Rhodococcus	2	0	2
	Kocuria	4	2	1
	Pseudoclavibacter	3	1	0
	Plantibacter	1	0	0
	Sanguibacter	1	0	0
	Pseudarthrobacter	1	0	0
	Microbacterium	4	1	1
	Salinibacterium	1	0	0
	Streptomyces	1	0	1
	Micrococcus	1	0	0
	Brachybacterium	1	1	0
	Clachiihabitans	1	0	0
	Microcella	1	0	0
Bacteroidetes	Flavobacterium	4	2	1
Total		167	41	73

Phylogenetic tree, based on 16S rRNA gene sequences (880 bp), showing the phylogenetic relationship between strains of the genera *Bacillus* and *Paenibacillus* isolated from epilithic biofilms in Lake Baikal. Accession numbers in GenBank are given in parentheses.

Table II

Antagonist activity (Zimens et al. 2014), and PKS and NRPS genes in the strains isolated from biofilms of Lake Baikal.

Strain	Antagonist activity	PCR signal to PKS gene	PCR signal to NRPS gene
Paenibacillus sp. 5A	Ec1^*, Ec2, Pa, Bs1, Bs2, Ca, Sa, Ef	+	+
Paenibacillus sp. 12A	Ec1, Ec2, Pa, Bs1, Bs2, Ca, Sa, Ef	+	+
Paenibacillus sp. 7A	Bs1, Bs2, Sa, Ca	+	+
Bacillus sp. 2A	Ec1, Bs1, Bs2, Ca	+	+
Bacillus sp. 9A	Bs1, Bs2, Ca, Sa, Ef	+	+
Bacillus sp. 2B	Ec1, Bs2	+	+

1* Test cultures used in this work: Bs1 – Bacillus subtilis VKPM; Bs2 – Bacillus subtilis DSM; Pa – Pseudomonas aeruginosa GISK L.A. Tarasevich 190158; Ca – Candida albicans ATCC 10231; Sa – Staphylococcus aureus (ATCC 25923 and MRSA); Ef – Enterococcus faecium; Ec1 – Escherichia coli K12 VKPM В-3254; Ec2 – Escherichia coli M17-02 VKPM В-8208

Table III

The strain screening for the presence of enzyme activity.

Characteristic	Paenibacillus spp.			Bacillus spp.
Characteristic	5A	12A	7A	2A	9A	2B
Phosphatase	–	–	–	+	+	+
Catalase	+	+	+	+	+	+
Oxidase	–	+	–	–	–	–
Gelatinase	–	–	–	+	+	–
Caseinase	+	+	+	+	+	+
Amylase	+	+	+	+	+	+
Lecithinase	+	+	–	+	+	+
Lipase	–	–	–	–	–	–
Saccharose	+	+	+	–	–	+
Glucose	+	+	+	–	–	+
Maltose	+	+	+	–	–	–
Fructose	+	+	+g^{^*}	–	–	+
Galactose	+g	+g	+	–	–	–
Lactose	+	+	+g	–	–	–
Arabinose	+	+	+	–	–	–
Raffinose	+	+	+	–	–	–
Rhamnose	+g	+g	+	–	–	–
Mannose	+	+	+	–	–	+
Xylose	+	+	+	–	–	–
Sorbitol	+	+	+	–	–	–
Inositol	+	+	+	–	–	+
Dulcite	+g	+g	+	–	–	–
Mannitol	+g	+g	+	–	–	–
Proline	–	–	+	+	+	+
Leucine	–	–	–	+	+	+
Phenylalanine	–	–	–	+	+	+
Alanine	–	–	–	+	+	+

1* +g – sugar fermentation together with the formation of acid and gas.

Phylogenetic tree, based on 16S rRNA gene sequences (1360 bp), showing the phylogenetic relationship between the strains studied and other species of the genera *Bacillus* and *Paenibacillus*. Sequences obtained in this study are in bold, and accession numbers in GenBank are given in parentheses.

Table IV

Comparative analysis of the sequences of PKS gene fragments from heterotrophic bacteria.

Strain name	Clone number	Results of BLAST analysis
Strain name	Clone number	Closest homologs	Homology, %
Paenibacillus spp. 5A and 12A	5A-1, 5A-2, 5A-3, 5A-4, 5A-7, 5A-8, 12A-2, 12A-9	ACN13122 ketosynthase [Streptomyces sp. G2-4]	99
		WP_053325747 polyketide synthase [P. peoriae]	99
		WP_013310977 polyketide synthase [P. polymyxa]	98–100
	5A-5,12A-1, 12A-5, 12A-6	ACX31707 ketosynthase [Streptomyces sp. 28HAO]	99
	5A-5,12A-1, 12A-5, 12A-6	WP_013310977 polyketide synthase [P. polymyxa]	99
	5A-6	WP_013310977 polyketide synthase [P. polymyxa]	97
	5A-6	WP_053325747 polyketide synthase [P. peoriae]	97
	12A-7, 12A-10	WP_053325746 polyketide synthase [P. peoriae]	97
	12A-7, 12A-10	WP_023989388 polyketide synthase [P. polymyxa]	97
	12A-8	WP_053325746 polyketide synthase [P. peoriae]	96
	12A-8	WP_023989388 polyketide synthase [P. polymyxa]	96
Paenibacillus sp.7A	7A-1, 7A-2, 7A-5, 7A-7 7A-3, 7A-4, 7A-6 7A-8	AIQ67612 erythronolid synthase [P. graminis]	96-98
		WP_042266339 NRPS/PKS-synthase [P. graminis]	98-99
		WP_042266418 polyketide synthase [P. graminis]	99
Bacillus spp. 2A and 2B	2A-1, 2A-3, 2A-4, 2A-6, 2A-7, 2A-8, 2B-3, 2B-4	ABR19768 polyketide synthase [B. subtilis]	98-99
	2A-2	ABR19764 polyketide synthase [Bacillus subtilis]	98
	2A-2	ABR19779 polyketide synthase [Actinomycetales bacterium DA20]	98
	2B-1	WP_032721576 polyketide synthase [B. subtilis]	97
	2B-1	AGA23985 NRPS/PKS-synthase [B. subtilis subsp. subtilis BSP1]	97
	2B-2	ABR19775 polyketide synthase [B. subtilis]	98
	2B-5	WP_043940121 polyketide synthase [Bacillus sp. YP1]	98
	2B-5	WP_009967299 polyketide synthase [B. subtilis]	98
	2B-6, 2B-8	ACG70843 polyketide synthase [Bacillus sp. WPhG3]	99
	2B-7	ABR19767 polyketide synthase [B. subtilis]	98
Bacillus sp. 9A	9A-1	AIO09652 ketosynthase [Bacillus sp. LX-110]	99
	9A-3, 9A-5	ACG70841 polyketide synthase [Bacillus sp. WPySW2]	98
	9A-3, 9A-5	AGL92430 polyketide synthase [B. amyloliquefaciens]	99
	9A-10	WP_016936042 polyketide synthase [B. siamensis]	99
	9A-10	WP_047474891 polyketide synthase [B. amyloliquefaciens]	99

Table V

Comparative analysis of the sequences of PKS genes from heterotrophic bacteria.

Strain	Clone number	Results of BLAST analysis
Strain	Clone number	Homologs with identified protein	Homology, %
Paenibacillus 5A and 12A	5A-1, 5A-2, 5A-3, 5A-4, 5A-7, 5A-8, 12A-2, 12A-9	EJD67453 difficidin synthase, (DfnD) [Bacillus sp. 916]	71
	5A-1, 5A-2, 5A-3, 5A-4, 5A-7, 5A-8, 12A-2, 12A-9	EIF13796 difficidin synthase, (DfnG) [Bacillus sp. 5B6]	71
	5A-5, 12A-1, 12A-5, 12A-6	BAP05593 calyculin synthase, (CalE), uncultured [Entotheonella sp.]	71
	5A-5, 12A-1, 12A-5, 12A-6	ADN68476 sorangicin synthase, (SorA) [Sorangium cellulosum So ce12]	71
	5A-6	WP_004619353 erythronolid synthase, [Clostridium papyrosolvens DSM 2782]	75
	5A-6	EIF13279 bacillaene synthase, (BaeL) [Bacillus sp. 5B6]	73
	12A-7, 12A-10	ADD82940 batumin synthase, (Bat2) [P. fluorescens BCCM_ID9359]	70
	12A-7, 12A-10	ABK51300 bryostatin synthase, (BryC) [Endobugula sertula]	69
	12A-8	EIF13279 bacillaene synthase, (BaeL) [Bacillus sp. 5B6]	72
	12A-8	ADN68477 sorangicin synthase, (SorB) [Sorangium cellulosum So ce12]	72
Paenibacillus sp.7A	7A-1, 7A-2, 7A-5, 7A-7	AIQ67612 erythronolid synthase, [P. graminis DSM 15220]	97
	7A-3, 7A-4, 7A-6	CUB31962 plipastatin synthase, [B. amyloliquefaciens]	53
	7A-3, 7A-4, 7A-6	WP004618786 erythronolid synthase, [Clostridium papyrosolvens DSM 2782]	50
	7A-8	WP_013663185 erythronolid synthase, [Marinomonas mediterranea MMB-1]	43
	7A-8	ADB12491 epothilone synthase, (EpoD) [Sorangium cellulosum KYC3013]	43
Bacillus spp. 2A and 2В	2A-1, 2A-3, 2A-4, 2A-6, 2A-7, 2A-8, 2В-3, 2В-4	EJD66458 bacillaene synthase, (BaeN) [Bacillus sp. 916]	85
	2A-2	BAP05593 calyculin synthase, (CalE), [Entotheonella sp.]	71
	2A-2	EJD67453 difficidin synthase, (DfnD) [Bacillus sp. 916]	70
	2B-1	EIF13279 bacillaene synthase, (BaeL) [Bacillus sp. 5B6]	85
	2B-2	EIF13280 bacillaene synthase, (BaeM) [Bacillus sp. 5B6]	84
	2B-5	EIF13279 bacillaene synthase, (BaeL) [Bacillus sp. 5B6]	71
	2B-6, 2B-8	EJD66458 bacillaene synthase, (BaeN) [Bacillus sp. 916].	87
	2B-7	EIF13279 bacillaene synthase, (BaeL) [Bacillus sp. 5B6].	86
	2B-7	AFZ90784 bacillaene synthase, (BaeL) [B. methylotrophicus AS43.3]	86
Bacillus sp. 9A	9A-1	WP_049628737 difficidin synthase, [Bacillus sp. JFL15]	98
	9A-3, 9A-5	EJD66458 bacillaene synthase, (BaeN) [Bacillus sp. 916]	94
	9A-10	EIF13279 bacillaene synthase, (BaeL) [Bacillus sp. 5B6]	97

Table VI

Comparative analysis of the sequences of NRPS genes from heterotrophic bacteria.

Strain	Clone number	Results of BLAST analysis
Strain	Clone number	Closest homologs	Homology, %
Paenibacillus spp. 5A and 12A	5A-4	WP_029514857 non-ribosomal peptide synthetase [P. polymyxa]	95
	5A-4	ADJ56349 fengycin A [Bacillus subtilis]	56
	5A-5, 12A-1, 12A-2	KOS00083 bacitracin synthetase [P. polymyxa]	97–100
	5A-5, 12A-1, 12A-2	ABQ96384 fusaricidin synthetase [P. polymyxa]	96–99
	12A-5	AKH45460 tridecaptin, TrbE [P. polymyxa]	96
	12A-5	KOS03006 fusaricidin synthetase [P. polymyxa]	96
	12A-6	WP_013310169 NRPS/PKS synthase [P. polymyxa]	96
		KOS01021 bacitracin synthetase [P. polymyxa]	94
		AHM66091 bacillorin synthetase b [P. polymyxa SQR-21]	94
		AKH45459 tridecaptin B, TrbD [P. polymyxa]	93
		KTS81295 fusaricidin synthetase [P. jamilae]	92
	12A-10	WP_025721680 non-ribosomal peptide synthetase [P. polymyxa]	99
		ABQ96384 fusaricidin synthetase [P. polymyxa]	96
		KOS01669 bacitracin synthetase [P. polymyxa]	94
Bacillus sp. 2B	2B-1	WP_080466694 plipastatin synthetase [B. subtilis]	100
Bacillus sp. 2B	2B-3	CAA49816 surfactin synthetase, srfA [B. subtilis]	99
Bacillus sp. 2A	2A-5	WP_019712401 plipastatin synthetase subunit B [B. subtilis]	98
	2A-5	ACX47457 fengycin synthetase B [B. subtilis].	98
	2A-7	AOA54360 polyketide synthase PksJ [B. subtilis]	98
	2A-7	WP_009967299 non-ribosomal peptide synthetase [B. subtilis]	99
	2A-8	WP_048654751 surfactin synthetase SrfAA [B. subtilis]	97
Bacillus sp. 9A	9A-1	WP_047474893 non-ribosomal peptide synthetase [B. amyloliquefaciens]	99
	9A-1	ASB53193 polyketide synthase PksJ [B. velezensis]	94
	9A-4	AHW81970 bacillomycin [B. subtilis]	96
	9A-8	WP_064777945 surfactin non-ribosomal peptide synthetase SrfAA [B. siamensis]	98
	9A-9	WP_045925809 non-ribosomal peptide synthetase [B. siamensis]	98
	9A-9	CCF05308 iturinA synthetase ItuA [B. velezensis CAU B946]	93

Neighbour-joining phylogenetic tree based on amino acid sequences of the KS-domain fragments of PKS genes in bacteria isolated from the epilithic biofilms of Lake Baikal (in bold). The scale bar represents 0.1 amino acid substitutions per site.

Neighbour-joining phylogenetic tree based on amino acid sequences of the A-domain fragments of NRPS genes in bacteria isolated from the epilithic biofilms of Lake Baikal (in bold): a) genus *Bacillus* and b) genus *Paenibacillus*. The scale bar represents 0.2 amino acid substitutions per site.

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DOI: https://doi.org/10.21307/pjm-2018-060 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331

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Language: English

Page range: 501 - 516

Submitted on: Jun 24, 2018

Accepted on: Oct 3, 2018

Published on: Dec 10, 2018

Published by: Polish Society of Microbiologists

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

polyketide synthase genes,

non-ribosomal peptide synthetases,

epilithic biofilms,

Lake Baikal,

secondary metabolites

Related subjects:

Life sciences,

Microbiology and virology

© 2018 ELENA SUKHANOVA, EKATERINA ZIMENS, OKSANA KALUZHNAYA, VALENTINA PARFENOVA, OLGA BELYKH, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 67 (2018): Issue 4 (December 2018)

Epilithic Biofilms in Lake Baikal: Screening and Diversity of PKS and NRPS Genes in the Genomes of Heterotrophic Bacteria

Figures & Tables

Table I

Fig. 1.

Table II

Table III

Fig. 2.

Table IV

Table V

Table VI

Fig. 3.

Fig. 4.

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