Bacterial diversity and metabolism in microbial consortium of non-axenic culture Tychonema sp. BBK16

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1. Introduction

Interactions occurring between algae and bacteria represent a particular circulation of organic matter (Azam et al., 1983). Cyanobacteria, as primary producers, form a specific habitat environment by forming organic matter and synthesizing biologically active metabolites (Woodhouse et al., 2018). Heterotrophic bacteria associated with cyanobacteria are more often species with flexible universal metabolism, most of the strains belong to Proteobacteria (Berg et al., 2009). High-throughput sequencing allows a detailed inventory of cyanobacteria-heterotrophic bacteria associations without cultivating (Shaw et al., 2020). For example, Nostoc macrocolony communities from lakes have been found to repeat partly a plankton-lake motif, but members of Shingomonadaceae, Rhodobacteriaceae, Comamonadaceae become distinctive (Aguilar et al., 2019). These bacteria are frequent companions of cyanobacteria in natural conditions (Chun et al., 2017; Park et al., 2021; Thorat et al., 2022). Metagenomic studies of the non-axenic cultures’ microbiome of subpolar cyanobacteria showed the occurrence of representatives of Proteobacteria and Bacteroidetes in the community and ruled out the possibility of external contamination confirming the co-evolution of cyanobacteria and their companions (Cornet et al., 2018). Non-axenic algal cultures are suitable model objects for studying interactions between algae and heterotrophic bacteria because they demonstrate in situ relationships. The presence of different bacteria in cyanobacterial cultures allows the study of their genomes using bioinformatics tools for nucleotide fragment separation (Tan et al., 2016).

The genome of the filamentous cyanobacterium Tychonema sp. BBK16 isolated from benthic biofilms revealed its ecology and genomic characteristics (Tikhonova et al., 2022; Evseev et al., 2023). Analysis of DNA isolated from a non-axenic culture of cyanobacterium revealed heterotrophic bacteria inhabiting the mucous cover during autotrophic biofilm growth in vitro. The aim of this work is to study the bacteria-companions of Tychonema sp. BBK16 and the metabolic characterization of this microbial consortium based on DNA barcoding and metagenomics data.

2. Material and methods

Sample collection and DNA sequencing

Obtaining a non-axenic culture of cyanobacterium and isolation of DNA was previously described (Tikhonova et al., 2022). The strain was cultured in vitro for seven years on autotrophic Z-8 medium containing nitrate, sulfate, carbonate, and phosphate as nutrients. Illumina Miseq sequencing of the V3-V4 region of 16S rRNA gene was performed according to the manufacturer`s instructions with primers 343F (CTCCTACGGRRSGCAG) and 806R (GGACTACNVVGGGTWTCTAAT) in Evrogen (Moscow). Shotgun sequencing was performed by different methods using the DNBSEQ-400 sequencer (MGI, China) by PCR-free protocol with enzymatic fragmentation (MGI, China) and using the Illumina MiSeq platform (Illumina, USA) by the paired-end reads in the case of MGI - 150 bp, in the case of Illumina - 300 bp. The quality of V3-V4 amplicon libraries and metagenomic sequencing results were assessed using the program MultiQC v. 1.12 (Ewels et al., 2016), and adapters were removed using the Trim Galore v. 0.6.5 program (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/, date last accessed: 12 December 2023). A phylogenetic tree was constructed using the BEAST v. l.8.4 (Drummond and Rambaut, 2007). Raw data deposited PRJNA1042932 (two metagenomic sequencing pools), SRR11929492 (16S rRNA gene sequencing data).

DNA metabarcoding data processing

The DADA2 v. 1.16 package for the R programming language was used for further processing, which included filtering out non-target and chimeric sequences, and clustering into ASVs (Amplicon Sequence Variants) (Callahan et al., 2016; R Core Team, 2021). Nearest homologues were selected by BLASTn searches using the NCBI nr/nt reference sequence databases. ASVs and nearest homologues were aligned using the ClustalO algorithm (Sievers et al., 2011).

Shotgun data processing

Assembly of reads into contigs was performed using the SPAdes v. 3.15.4 (Prjibelski et al., 2020). Mapping of reads to the produced contigs was performed with BWA v. 0.7.17 and the Samtools v. 1.18 package (Heng and Durbin, 2009; Danecek et al., 2021). Metagenomic binning and genome isolation was performed using MetaBAT2 v. 2.15 (Kang et al., 2019). Completeness and contamination of the final MAGs were evaluated using CheckM2 v. 1.0.2 based on a machine learning algorithm (Chklovski et al., 2022).

Open reading frames (ORFs) in contigs were detected using Prodigal v. 2.6.3 (Hyatt et al., 2010). KEGG annotation and assignment of KO numbers to proteins were performed using the BlastKOALA service (Kanehisa et al., 2016) and semi-automatically using DIAMOND v. 2.1.8 (Buchfink et al., 2021) against the NCBI nr protein sequence database. Ribosomal RNA genes were isolated using Barrnap v. 0.9 (Seemann, 2013). Taxonomic annotation of contigs and MAGs was performed using the Kaiju service (Menzel et al., 2016) and manually improved using author scripts based on metabolic annotation obtained previously.

The functional characteristics of microorganisms were established based on the presence of marker genes for metabolic processes in the genome, similar to the methodology described by Garner et al. (2023). Carbon autotrophy was established by the presence of genes in the CBB cycle - rbcL, prkB; glyoxylate assimilation pathway - mct; ammonia transport into the cell - amt, and its assimilation - glnA; transport of nitrite/nitrate - nrtABC, assimilation of nitrates/nitrites - narB, nirA, nasABED; denitrification genes - narGHI, nirK, norB, nosZ; urea transport - urtABCDE and its decomposition to ammonia - ureABC; carboxylation of urea - E6.3.4.6 urea carboxylase; ammonium production from glutamate – asnB; phosphonate transport phnCDE; phosphonate decomposition – phnAB; decomposition and modification of phosphonates - phnIJKLMPWXY; assimilation of phosphate from organic compounds – phoD; transport of inorganic phosphate - pstBS; hydrolytic enzymes for polysaccharides - argH, glgX, susACD; sulfate and thiosulphate transport system - cysAPUW; oxidation of sulfates and thiosulphates in periplasm - soxABCDXZ; degradation of aromatic compounds - vanA, dmpB, xylE; assimilation of carbon monoxide with the release of hydrogen - coxMLS, cutML; permeases for facilitated transport of oligopeptides and oligosaccharides - oppABCDF, mppA; methylamine utilisation (methylotrophy)- gmaS, mgsABC, mgdABCD; use of carbon monoxide - coxMLS, cutML; synthesis of bacteriochlorophylls - bchMO; light-gathering polypeptides, photosystem II - pufML.

3. Results and discussion

Phylogenetic diversity and bacterial genomes in a non-axenic culture Tychonema sp. BBK16

Sequencing of the V3-V4 amplicon library of 16S rRNA gene resulted in 62,028 paired-end reads. From this set, 21 ASVs were isolated, the annotation of which revealed representatives of three phyla: Cyanobacteria (66.1%), Pseudomonadota/Proteobacteria (33.7%), and Actinobacteriota (0.2%). The most represented genera are Hydrogenophaga, Sphingomonas, Paucibacter, Pseudomonas, Aminobacter, Devosia, Tahibacter, Bosea, Methylophilus, Rhodopseudomonas, Ensifer, Tabrizicola, Acidovorax, Caulobacter; minor genera are Rhodococcus and Iamia (Table 1).

 

Table 1. Abundance of ASVs in the biofilm of Tychonema sp. BBK16 sample

ID	Phylum	Class	Genus	Count	Value, %
ASV001	Cyanobacteriota	Cyanobacteria	Tychonema CCAP 1459-11B	41016	66.12
ASV003	Pseudomonadota/Proteobacteria	Betaproteobacteria	Hydrogenophaga	10959	17.66
ASV005	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Sphingomonas	5116	8.24
ASV012	Pseudomonadota/Proteobacteria	Betaproteobacteria	Paucibacter	1041	1.67
ASV009	Pseudomonadota/Proteobacteria	Gammaproteobacteria	Pseudomonas	840	1.35
ASV016	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Aminobacter	789	1.27
ASV017	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Devosia	669	1.08
ASV004	Pseudomonadota/Proteobacteria	Gammaproteobacteria	Tahibacter	635	1.02
ASV020	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Devosia	433	0.7
ASV037	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Devosia	147	0.24
ASV041	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Bosea	81	0.13
ASV047	Actinobacteriota	Actinobacteria	Rhodococcus	71	0.11
ASV049	Pseudomonadota/Proteobacteria	Gammaproteobacteria	Methylophilus	60	0.01
ASV050	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Rhodopseudomonas	58	0.094
ASV051	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Ensifer	52	0.084
ASV056	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Tabrizicola	36	0.058
ASV027	Actinobacteriota	Actinobacteria	Rhodococcus	14	0.023
ASV093	Pseudomonadota/Proteobacteria	Gammaproteobacteria	Acidovorax	3	0.005
ASV092	Pseudomonadota/Proteobacteria	Alphaproteobacteria	Caulobacter	3	0.005
ASV091	Actinobacteriota	Acidimicrobiia	Iamia	3	0.005
ASV095	Proteobacteria	Gammaproteobacteria	Acidovorax	2	0.003

 

Shotgun sequencing generated 18,4421,914 paired-end reads; taxonomic annotation of which revealed representatives of three phyla: Cyanobacteria (50.2%), Proteobacteria (49.6%), and Actinobacteriota (0.2%). Metagenomic binning of the contigs obtained after assembly identified 12 bacterial genomes of different quality (Table 2). The most complete genomes according to CheckM2 completeness and contamination statistics (given in parentheses, %), were the following: Tychonema sp. BBK16 (bin.4 — 99.15 / 2.42), Tahibacter sp. (bin.3 — 99.3 / 0.09), Aminobacter sp. (bin.8 — 97.44 / 1.83), Paucibacter sp. (bin.9 — 100 / 0.26), Sphingomonas sp. (bin.11 — 99.76 / 8.07). Also noteworthy is the high percentage of assembly of the Devosia, Bosea, and Hydrogenophaga genomes.

 

Table 2. Metagenome-assembled genomes isolated from the Tychonema sp. BBK16 biofilm sample. Bins with >50% completeness and <5% contamination are in bold.

bin ID	Compl. *	Cont.*	Contig N50	Genome Size	GC Content	Total CDS	Genus
bin.1	20.8	0.35	3434	1190380	0.63	1405	Rhodococcus
bin.2	7.19	0.01	149685	412532	0.6	443	Aminobacter
bin.3	99.3	0.09	214065	6110546	0.66	4716	Tahibacter
bin.4	99.15	2.42	108947	5876576	0.44	5150	Tychonema
bin.5	65.82	0.01	39311	2077122	0.69	1999	Hydrogenophaga
bin.6	84.19	2.56	14881	4250091	0.64	4320	Devosia
bin.8	97.44	1.83	134644	5591995	0.63	5427	Aminobacter
bin.9	100	0.26	214794	4356207	0.67	3973	Paucibacter
bin.10	84.96	3.7	10883	5032618	0.66	5129	Bosea
bin.11	99.76	8.07	90952	3846091	0.69	3710	Sphingomonas
bin.12	15.11	0.04	3530	797961	0.67	888	Stenotrophomonas

Note: * Compl. – Completeness, Cont. - Contamination

 

To assess the convergence of the targeting and metagenomic sequencing results, a phylogenetic tree was constructed based on the DNA alignment of the fragment 16S rRNA gene (Fig. 1). According to the BLASTn search, the tree also contains the nearest homologues. As shown in the figure, in most cases, it is possible to find a pair of sequences from ASVs and metagenomes belonging to the same genus/species that indicate sufficient sequencing depth for both methods. Even when no 16S rRNA marker gene was detected in the metagenome, other coding sequences and genome fragments were annotated as taxa represented in the tree.

 

Fig.1. Phylogenetic tree based on DNA alignment of the fragment of the 16S rRNA gene. ASVs obtained from DNA-metabarcoding data are highlighted in red, fragments of 16S rRNA gene from the metagenome are in blue, and nearest neighbors by BLASTn-search are in black color

 

Metabolic functions of bacteria in the Tychonema sp. BBK16 biofilm

According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, a total of 2387 different functional orthologs (KO) were identified in the biofilm-consortium. We presented 8 bins to describe the bacterial functions (Fig.2). The analysis of the main pathways of carbon metabolism showed that the most significant participants in the biofilm community are the metabolism of carbohydrates and amino acids; a large part is represented by the categories of signaling and cellular processes, nucleotide and energy metabolism, membrane transport, and the metabolism of cofactors and vitamins. See Supplementary material (S1) for additional analysis - comparison of predicted functions of microorganisms and actual functional genes.

 

Fig.2. Abundance heatmap of different metabolic processes in the biofilm of Tychonema sp. BBK16

 

Functional annotation of the assembled genomes identified key metabolic markers of biofilm community members (Table 3). The ability to fix inorganic carbon was confirmed by the presence of the enzymes of the CBB cycle in the microorganisms Tychonema sp. BBK16 and Hydrogenophaga sp., although in the latter this process is facultative and occurs when there is a deficit of organic matter in the medium. The ability to use solar energy is confirmed by the presence of bacteriochlorophyll, carotenoid synthesis genes, and photosystem II reaction center enzymes in the bacterium Bosea sp.

 

Table 3. Microbial processes in the biofilm of Tychonema sp. BBK16

Bacterium	Metabolism	Confirmative genes
Tychonema sp. BBK16	Autotroph. Uses nitrate, nitrite, ammonia and urea. Assimilates phosphates of inorganic and organic compounds, synthesizes alkaline phosphatase. Utilizes sulfates and thiosulphates.	rbcL, prkB, amt, nrtA, narB, nirA, glnA, ureABC , urtABCE, pstB, pstS phoD, cysU, cysW
Tahibacter sp.	Organotroph. Uses phosphates, phosphonates, and ammonia, hydrolyses glycans. Denitrification.	argH, amt, phnA, pstBS, nirK, norB
Hydrogenophaga sp.	Chemoorganotroph/chemolithoautotroph. Oxidises hydrogen as a source of energy and CO2 or simple organic matter as a carbon source. Preferred nitrogen source is urea, glutamate. Able to degrade oligosaccharides. Transports nitrate and phosphate. Participates in thiosulfate oxidation by the periplasmic SOX enzyme complex.	rbcL, prkB, soxABCDXZ, urtABCE ureABC, urea carboxylase, amt, asnB, cysU, cysW
Devosia sp.	Organotroph. Utilizes organic and inorganic sources of nitrogen and phosphorus, detoxifies urea by decomposition or incorporation into organic compounds. Contains a large number of transporters of oligopeptides and simple organic matter, modifies aromatic compounds. Possesses a powerful chemotaxis system.	glgX, nasC, nasABED, nrtABC, oppABCDF, mppA, phnСIJMP, pstBS, urtAE, ureaABC, urea carboxylase, vanA
Aminobacter sp.	Organotroph. Mobile due to piles, hydrolyses polysaccharides, catechols, glycogen, urea, assimilates nitrate, phosphate, sulfate, thiosulphate, denitrifier. Oxidizes carbon monoxide to carbon dioxide. Methylotroph involved in the degradation of methylamines (serine pathway).	amt, argH, glgX, dmpB, xylE, nasABED, nosZ, nrtABC, pstBS, phnACDIJKLMPWY; soxABG, cysU, cysW, urtABCE, ureABC, gmaS, mgsABC, mgdABCD, coxMLS, cutML
Paucibacter sp.	Aerobic heterotroph capable of hydrolysing complex polysaccharides, a companion bacterium to cyanobacteria in nature and cultures, utilizes nitrate, phosphate, sulfate, thiosulphate. Possible denitrification.	amt, argH, narGHI, nasABED, nrtABC, phoD, pstBS, phnDEX, soxBCDXZ, cysU, cysW, susA, coxMLS, cutML
Bosea sp.	Anoxygenic aerobic phototroph, contains bacteriochlorophyll a and photosystem II. Assimilates nitrate and phosphate.	amt, glgX, argH, bchM, chlM, bchO, bchX,Y,Z, pufM,L, nrtAC, nasDF, urtABC, pstBS, phnCIJKMPXY
Sphingomonas sp.	Universal organotroph capable of carbon autotrophy, fixes carbon dioxide using an alternative carbon pathway, the glyoxylate cycle. Hydrolyses polysaccharides. Assimilates phosphates and phosphonates, produces alkaline phosphatase for phosphorus production from organic compounds.	amt, argH, mct, phoD, pstBS

 

A complex cycle of biogenic elements – phosphorus, nitrogen, and sulfur – occurs in the biofilm. Thus, the source of phosphorus is phosphates, which in a closed ecosystem are produced by the activity of the enzyme alkaline phosphatase of the microorganisms Tychonema, Paucibacter, and Sphingomonas. Most bacteria have phosphonate transport systems. These organic phosphorus compounds are common in natural ecosystems and were once the first sources of phosphorus for ancient microorganisms (McGrath et al., 2013). Phosphonate transport systems can also serve as phosphate transporters (Stasi et al., 2019).

Traditionally, the relationship between autotroph and heterotrophic bacteria is considered as a metabolic symbiosis “carbon in exchange for nitrogen” implying that bacteria fix atmospheric nitrogen. Thus, a microbial community represented by the filamentous cyanobacterium Microcoleus vaginatus and strains of Actinobacteria and Proteobacteria, many of which were atmospheric nitrogen fixers, was isolated from arid soils (Nelson et al., 2021). In our case, nitrogen fixation was not detected, which was confirmed by the predictive method (PICRUSt2) and the absence of marker genes for the process in the genome (Supplementary materials, S2, S3). Genes responsible for denitrification (nitrogen removal) were identified in three members of a consortium related to Proteobacteria (Aminobacter, Tahibacter, and probably Paucibacter). According to genomic characteristics, the main sources of nitrogen for all members of the consortium may be ammonium, nitrate, nitrite, and urea (as a metabolite and as a decomposition product of dead biomass). Genes related to ammonium transport and ammonium assimilation enzymes were detected in all consortium members. Genes coding for urea transport and its degradation to ammonium were revealed in cyanobacteria and heterotrophic bacteria, except Sphingomonas, Paucibacter, and Tahibacter.

Thus, all metabolic processes are necessary to keep the whole community alive. The autotrophic cyanobacterium Tychonema sp. BBK16, as well as the facultative autotrophs Hydrogenophaga and Sphingomonas, forms organic matter for bacteria. However, it has genes for organic matter assimilation being a mixotroph (Evseev et al., 2023). Facultative autotrophy of hydrogen-oxidizing bacteria is an inducible process; they are successful organotrophs in the presence of simple organic matter (Zavarzin, 1972). Bacteria Aminobacter, Tahibacter, Devosia, and Paucibacter are able to consume polysaccharide substances, which are abundant in cyanobacterial biofilm. Aminobacter has the most extensive metabolism being an active polysaccharide degrader, methylotroph, denitrifier, and, as well as Paucibacter, a supplier of hydrogen for the hydrogen-oxidizing bacteria. Hydrogen is also released during the anoxygenic photosynthesis of the bacterium Bosea. Devosia are able to inhabit places rich in organic compounds, such as wastewater and biofilms, due to transport proteins - permeases, which uptake short peptides of various amino acid compositions serving as a source of carbon and nitrogen (Talwar et al., 2020). During the phosphate depletion period, Sphingomonas, Tychonema, and Paucibacter additionally produce alkaline phosphatase to hydrolyze phosphate-containing organic matter. Cyanobacteria Tychonema sp. BBK16 is dependent on other bacteria because the system is closed and nitrate and nitrite cannot be provided from outside, while ammonium is released by the bacterial decomposition of nitrogen-containing polysaccharides and glycosides, proteins and amino acids.

It was previously suggested that the microbiome associated with cyanobacteria represents an “ecological footprint” of the habitat (Cornet et al., 2018). We assume that the microbial consortium studied with Tychonema is represented by typical inhabitants of substrates rich in organic and has all necessary for this purpose: enzymes of denitrification, methylotrophy, hydrolysis of aromatic compounds and complex polysaccharides, as well as organic compounds of nitrogen, phosphorus, and sulfur. Some of the genera have been described such as Hydrogenophaga, Methylophilus and Pseudomonas are found in the cultivated diatoms of Lake Baikal (Mikhailov et al., 2018). In the article also showed that the Nocardioides are satellites of Baikal cultivated diatom algae.

4. Conclusion

We have demonstrated that Proteobacteria are the main symbionts of the filamentous cyanobacterium Tychonema sp. BBK16 in vitro. The dominant genera were Hydrogenophaga, Sphingomonas, Paucibacter, Aminobacter, Devosia, and Tahibacter. The studied biofilm community showed processes of oxygenic and anoxygenic photosynthesis (phototrophy and photoheterotrophy), facultative carbon dioxide fixation involving the glyoxylate pathway and SBB, methylotrophy, degradation of polysaccharides and aromatic compounds to oligosaccharides, organic acids, aldehydes, peptides, and monosaccharides, and organic polymers of nitrogen and phosphorus.

Acknowledgments

The authors are grateful to Y.S. Bukin, O.N. Pavlova, S.V. Bukin, I.S. Mikhailov, Y.R. Zakharova for their valuable discussions and help. Metagenomic sequencing using DNA nanoball sequencing (MGI) technology was supported by Helicon Company (Moscow). The research is funded by State Project 0279-2021-0015, “Viral and bacterial communities as the basis for the stable functioning of freshwater ecosystems...”.

Conflicts of Interest

The authors declare no conflicts of interests.

Sobre autores

A. Krasnopeev

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Autor responsável pela correspondência
Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

I. Tikhonova

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

G. Podlesnaya

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

S. Potapov

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

A. Gladkikh

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

M. Suslova

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

I. Lipko

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

E. Sorokovikova

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

O. Belykh

Limnological Institute of the Siberian Branch of the Russian Academy of Sciences

Email: krasnopeev@lin.irk.ru
Rússia, Ulan-Batorskaya Str., 3, Irkutsk, 664033

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