Genomic analysis of a pure culture of magnetotactic bacterium Terasakiella sp. SH-1*

Haijian DU , Wenyan ZHANG , Wei LIN , Hongmiao PAN ,Tian XIAO ,**, Long-Fei WU

1 College of Life Science, Shandong University, Qingdao 266237, China

2 CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences,Qingdao 266071, China

3 Laboratory for Marine Ecology and Environmental Science, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China

4 Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China

5 Key Laboratory of Earth and Planetary Physics, Institute of Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, China

6 Institutions of Earth Science, Chinese Academy of Sciences, Beijing 100029, China

7 France-China Joint Laboratory for Evolution and Development of Magnetotactic Multicellular Organisms, Chinese Academy of Sciences, Beijing 100029, China

8 Aix-Marseille University, CNRS, LCB, Marseille F-13402, France

Abstract Magnetotactic bacteria (MTB) display magnetotaxis ability because of biomineralization of intracellular nanometer-sized, membrane-bound organelles termed magnetosomes. Despite having been discovered more than half a century, only a few representatives of MTB have been isolated and cultured in the laboratory. In this study, we report the genomic characterization of a novel marine magnetotactic spirillum strain SH-1 belonging to the genus Terasakiella that was recently isolated. A gene encoding haloalkane dehalogenase, which is involved in the degradation of chlorocyclohexane, chlorobenzene, chloroalkane, and chloroalkene, was identif ied. SH-1 genome contained cysCHI and soxBAZYX genes, thus potentially capable of assimilatory sulfate reduction to H 2 S and using thiosulfate as electron donors and oxidizing it to sulfate.Genome of SH-1 also contained genes encoding periplasmic dissimilatory nitrate reductases ( napAB),assimilatory nitrate reductase ( nasA) and assimilatory nitrite reductases ( nasB), suggesting that it is capable of gaining energy by converting nitrate to ammonia. The pure culture of Terasakiella sp. SH-1 together with its genomic results off ers new opportunities to examine biology, physiology, and biomineralization mechanisms of MTB.

Keyword: magnetotactic bacteria; magnetotaxis; pure culture; comparative genomic analysis

1 INTRODUCTION

Magnetotactic bacteria (MTB) are prokaryotes that orient and migrate along the geomagnetic f ield lines,a behavior referred to as magnetotaxis or microbial magnetoreception. MTB share the capacity to synthesize magnetosomes, which are magnetic crystals of magnetite (Fe3O4) and/or greigite (Fe3S4)and enveloped by a phospholipid bilayer membrane(Blakemore, 1982; Bazylinski et al., 1995; Bazylinski and Frankel, 2004). Many morphotypes of MTB,including coccoid, ovoid, rod, vibrio, spirillum, and multicellular forms have been observed worldwide across from freshwater, brackish, and marine waters to waterlogged soils (Maratea and Blakemore, 1981;Hanzlik et al., 2002; Sakaguchi et al., 2002; Lefèvre et al., 2009; Wenter et al., 2009; Zhou et al., 2013; Lin and Pan, 2015; Lin et al., 2017a).

All known MTB belong to the domain Bacteria,predominantly within the phylum Proteobacteria(Simmons et al., 2004; Jogler and Schüler, 2009;Lefèvre and Bazylinski, 2013; Ji et al., 2017; Lin et al., 2018). A diverse group of MTB affi liated with the phylum Nitrospirae, the candidate phylum Omnitrophica, the candidate phylum Latescibacteria,and the phylum Planctomycetes have been identif ied(Vali et al., 1987; Lefèvre et al., 2010; Kolinko et al.,2012; Lin et al., 2018). More recently, our knowledge of the phylogenetic diversity of MTB has been dramatically expanded and so far, these organisms have been found in up to 16 bacterial phylum-level lineages (Lin et al., 2020; Uzun et al., 2020). Most of the cultured MTB belong to the phylum Proteobacteria, and a large number of these are within the Alphaproteobacteria, such as freshwaterMagnetospirillumspecies MS-1, AMB-1, MSR-1,and XM-1 (Blakemore et al., 1979; Matsunaga et al.,1991; Schleifer et al., 1991; Wang et al., 2016),marine vibrio strains MV-1 and MV-2 (Delong et al.,1993; Bazylinski et al., 2013), marine spirillum strains MMS-1 and QH-2 (Zhu et al., 2010; Williams et al., 2012), and recently isolated marine spirillumCandidatusTerasakiella magnetica strain PR-1 belonging to the genusTerasakiella(Monteil et al.,2018).

Much genomic data on MTB has now been obtained and analyzed. The whole genomes of some cultured MTB (e.g., AMB-1, MSR-1, QH-2, MC-1,MO-1, XM-1, BW-2, and SS-5) have been obtained(Matsunaga et al., 2005; Richter et al., 2007; Schübbe et al., 2009; Ji et al., 2014; Wang et al., 2016; Ji et al.,2017; Uebe et al., 2018; Geurink et al., 2020;Trubitsyn et al., 2021). While many other MTB remain to be draft sequenced. After decades of research on MTB genomes, many interesting features have been revealed. In particular amongst these is the presence of clustered genes that control magnetosome biomineralization and arrangement in MTB cells,which are termed magnetosome gene islands (MAIs)(Grünberg et al., 2001; Murat et al., 2010; Lohße et al., 2011) or magnetosome gene clusters (MGCs) (Lin et al., 2017b).

We recently isolated a new marine magnetotactic spirillum (designatedTerasakiellasp. SH-1) into axenic culture from an intertidal zone in Sanya, China(Du et al., 2019). SH-1 belongs to the genusTerasakiellain the Alphaproteobacteria and is closely related toCa. Terasakiella magnetica strain PR-1(Monteil et al., 2018). In the previous study, we have reported a duplication event of magnetosome genes withinmamABoperon in the genome of SH-1, which suggests that gene duplication event plays a potentially important role in the evolution of magnetotaxis in the Alphaproteobacteria (Du et al., 2019). Here, we perform a comparative analysis of the complete genome of SH-1 with representative MTB genomes,which provides novel insights in to metabolic potential and ecosystem function of this newly isolated MTB strains.

2 MATERIAL AND METHOD

2.1 Genome analysis

The whole-genome sequencing of the SH-1 was performed as previously described (Du et al., 2019).All genome data used in this study was downloaded from the NCBI site. The gene prediction was performed on the MicroScope platform (Vallenet et al., 2013). The tandem repeats annotation was obtained using the Tandem Repeat Finder (http://tandem.bu.edu/trf/trf.html) (Benson, 1999), and the minisatellite DNA and microsatellite DNA were selected based on the number and length of repeat units. Prophage regions were predicted using the PHAge Search Tool Enhanced Release (PHASTER)web server (http://phaster.ca/) (Arndt et al., 2016) and CRISPR identif ication using CRISPRFinder (Grissa et al., 2007). The best hit was performed using the BLAST (Altschul et al., 1990) alignment tool for function annotation. Seven databases including Kyoto Encyclopedia of Genes and Genomes (KEGG)(Kanehisa et al., 2016), Clusters of Orthologous Groups (COG) (Galperin et al., 2015; Makarova et al., 2015), Non-Redundant Protein Database databases(NR), Swiss-Prot (Consortium, 2015), Gene Ontology(GO) (Ashburner et al., 2000), TrEMBL (Apweiler et al., 2004), and EggNOG (Huerta-Cepas et al., 2016)were used for general function annotation.

2.2 Nitrogen metabolism analysis

Protein sequence similarities in relation to nitrogen metabolism were determined using BLAST on the MicroScope (Vallenet et al., 2013) platforms. Proteins in diff erent organisms were def ined as orthologs when their alignments met the criteria: E-value <1e-5,identity >30%, and query coverage >50%. The gene markers used to decide whether particular transformation reactions were present were based ona published review (Kuypers et al., 2018)(Supplementary Table S1); the markers were for genes involved in 15 reactions involving eight key inorganic nitrogen species having diff erent oxidation states.

Table 1 General features of Alphaproteobacteria MTB genomes of SH-1, PR-1, MV-1, QH-2, AMB-1, and MSR-1

2.3 Pan/core genome analysis

A pan/core genome analysis using the MicroScope(Vallenet et al., 2013) gene families (MICFAM),computed using the SiLiX software (Miele et al.,2011) (threshold: 50% of amino-acid identity and 80% of alignment coverage).

3 RESULT AND DISCUSSION

3.1 Genome overview of SH-1

The genome of SH-1 comprised 3 832 570 bp in a circular chromosome having the average G+C content of 47.5%. The chromosome contained 3 664 predicted coding sequences (CDS), including those encoding 52 tRNAs and three sets of rRNAs (5S, 16S, and 23S), which corresponded to 90.7% of the genome being coding sequences. Compared to freshwaterM.magneticumAMB-1 andM.gryphiswaldenseMSR-1, the genome size, G+C content, and CDS number of marine magnetospirilla (SH-1, PR-1, MV-1, and QH-2) are smaller (Table 1). The G+C content of SH-1 is smaller than those of MV-1 and QH-2 but higher than that of PR-1 (Table 1). In particular, SH-1 has a higher number of tRNAs and rRNA operons(Table 1). There was no evidence for the presence of extrachromosomal elements such as plasmids for SH-1. A total of 1 847 CDS (50.8%) could be assigned to putative functions, 1 227 CDS (33.8%) represented conserved hypothetical proteins of unknown function,and the remaining 559 CDS (18.4%) show no sequence similarity to any previously reported sequence. We identif ied 76 tandem repeats in the genome of SH-1, including 51 minisatellite DNAs and four microsatellite DNAs. No CRISPR sequence was found. In addition, three prophages were identif ied to be distributed throughout the genome.

3.2 Carbon metabolism

Genes involved in glycolysis (core module involving three-carbon compounds), the tricarboxylic acid (TCA) cycle, the non-oxidative phase of reductive pentose phosphate pathway, phosphoribosyl diphosphate (PRPP) biosynthesis, the ethylmalonyl pathway, and the synthesis of all 20 essential amino acids were identif ied in SH-1 genome (Fig.1). Genes involved in the Embden-Meyerhof pathway in glycolysis including hexokinase (enzyme commission(EC): 2.7.1.1), glucokinase (EC: 2.7.1.2), and glyceraldehyde-3-phosphate dehydrogenase (EC:1.2.7.6 and EC: 1.2.1.9) were missing. Most of the genes involved in reductive tricarboxylic acid (rTCA)cycle were detected, except for ATP-citrate lyase (EC:2.3.3.8), suggesting that SH-1 is not able to use the rTCA cycle for autotrophy.

Protein encoded by gene SH1_v1_2542 was found to be 67.1% identical to a fragment (170 aa/310 aa) of the genedhlAencoding haloalkane dehalogenase(EC: 3.8.1.5), which was previously identif ied and characterized inXanthobacterautotrophicusinvolved in the degradation of chlorocyclohexane,chlorobenzene, chloroalkane, and chloroalkene(Fig.1). Organisms having haloalkane dehalogenase are able to degrade hexachlorocyclohexane to 2,3,4,5,6-pentachlorocyclohexanol and 2,3,5,6-tetrachloro-1,4-cyclohexanol, and to degrade 1.2-dichloroethane to glyoxylate (Janssen et al.,1989). Similar genes have not been detected in the genomes of other MTB or other organisms belonging to genusTerasakiella. Its presence in SH-1 could be a result of horizontal gene transfer (HGT). It should be noted that active-site residues His289and Asp260of DHLA are not present in SH1_v1_2542, thus its function awaits further detailed characterization(Supplementary Fig.S1) (Verschueren et al., 1993).Furthermore, additional studies are needed to assess whether SH-1 has the ability to degrade chlorocyclohexane, chlorobenzene, chloroalkane,and chloroalkene, and to investigate the broader complexity of carbon metabolism by SH-1.

Fig.1 Reconstruction of metabolic pathways in SH-1

3.3 Sulfur metabolism

Unlike AMB-1, MSR-1, and QH-2, no evidence was found in the genome of SH-1 for the presence of any of the genes for dissimilatory sulfate reduction,includingdsrgenes or the Apr system. However,cysNandcysDwere present in the genome of SH-1, which encode enzymes that activate sulfate and catalyze the synthesis of adenosine-5′-phosphosulfate (APS) in the assimilatory sulfate reduction pathway (Fig.1). Genes ofcysC(APS kinase; produces 3′-phosphoadenosine-5′-phosphosulfate: PAPS),cysH(PAPS reductase;catalyzes the conversion of PAPS to sulf ite), andcysI(sulf ite reductase) were also identif ied. Therefore,SH-1 may be capable of assimilatory sulfate reduction to H2S (Pinto et al., 2004). SH-1 also containedsoxBAZYXgene cluster involved in thiosulfate oxidation, and thesoxCDgenes encoding sulfur dehydrogenase were also found (Fig.1). These f indings suggest that SH-1 could use thiosulfate as an electron donor and oxidize it to sulfate (Hensen et al., 2006).

3.4 Nitrogen metabolism

Both SH-1 and PR-1 contained genes encoding periplasmic dissimilatory nitrate reductases (napAB),assimilatory nitrate reductase (nasAB), hydroxylamine reductase (hcp), and assimilatory nitrite reductases(nasB) (Fig.1), suggesting that they can gain energy by converting nitrate to ammonia (Kuypers et al.,2018).nirBis identif ied in genomes of SH-1 and PR-1, butnirDis not present. Whether NirB alone could carry out the function needs more studies.napABare present in the genomes ofTerasakiellapusillabutnasABorhcpare not identif ied (Fig.2c & d). However,T.pusillacontains genes encoding cytochrome c-dependent nitric oxide (NO) reductases (cNOR andcnorB), and nitrous oxide reductases (nosZ) (Fig.2c),which are not present in SH-1 and PR-1. This may ref lect diff erences in the metabolism of MTB compared with other bacteria belonging to the same genus. Although genesf ixS,f ixG, andf ixIwere identif ied, thenifgene cluster responsible for nitrogen f ixation was not present in the genomes of genusTerasakiella. Whether the genusTerasakiellacan f ix nitrogen needs further investigation.

Fig.2 Microbial transformations of nitrogen compounds (modif ied from Kuypers et al., 2018)

We further compared the microbial transformations of nitrogen among 14 reprentative species of MTB(Terasakiellasp. SH-1,CandidatusTerasakiella magnetica strain PR-1,Magnetospirasp. strain QH-2,Magnetospirillumgryphiswaldensestrain MSR-1,Magnetospirillummagneticumstrain AMB-1,MagnetococcusmarinusMC-1, Magneto-ovoid bacterium MO-1,Ectothiorhodospiraceaebacterium BW-2, Gammaproteobacteria magnetotactic strain SS-5,DesulfovibriomagneticusRS-1,CandidatusMagnetomorum sp. HK-1,CandidatusMagnetoglobus multicellularis str. Araruama,CandidatusMagnetobacterium bavaricum andCandidatusMagnetoovum chiemensis) (Fig.2a & b). Seven reactions have been found in one or more genomes of the tested microorganisms (Fig.2b), including nitrate reductase (nasA,narGH, andnapA), heme-containing nitrite reductases (nirS), nitric oxide reductase (hcp,cnorB, andnorVW), nitrous oxide reductase (nosZ),assimilatory (nasBandnirBD) and dissimilatory nitrite reductase (nrfAHand OTR), nitrogenases(nifHDK), cyanase (cynS), and urease (ureABC).

Among these enzymes, genes encoding for nitrate reductases, nitrite reductase and nitric oxide reductases were most commonly found. These genes occur widely in genomes of Alphaproteobacteria,Gammaproteobacteria, and Etaproteobacteria. Our results show that MTB commonly act as denitrif iers and nitrogen-f ixers in nitrogen cycle processes.Previous studies have shown the potential link between denitrif ication and magnetosome formation in freshwaterMagnetospirillumspp. (Bazylinski and Blakemore, 1983; Matsunaga et al., 1991; Matsunaga and Tsujimura, 1993; Yang et al., 2001; Li et al.,2012). Therefore, denitrif ication should play an important role in redox control for magnetosome formation. However, their role in magnetosome formation has not yet been fully clarif ied and needs further experimental investigation.

3.5 Prophage

Two predicted prophage regions were found in the genome of SH-1, having lengths of 11.0- 22.6 kb and G+C contents of 46.1%- 48.4% (Supplementary Table S2 & Fig.S2). Among these, SH1_v1_1640- 1665 comprised 23 CDS containing three predicted phage tail collar domain proteins, two plasmid maintenance system killer proteins (HigB),and seven transposases, one sulfotransferase and seven hypothetical proteins (Supplementary Fig.S2a& b1), while SH1_v1_2382 to SH1_v1_2398 comprised 16 CDS, including three putative tailrelated proteins, one putative endolysin, one DNA maturase beta subunit, one putative major capsid protein, and one putative GcrA-like cell cycle regulator (Supplementary Fig.S2a & 2b). No genes encoding lysine and holin, representing the lysis module, were found.

Fig.3 Venn diagram of pan/core gene analysis in the genus Terasakiella

We searched genomes of 12 bacteria (Terasakiellasp. SH-1,CandidatusTerasakiella magnetica strain PR-1,Magnetospirasp. strain QH-2,Magnetospirillumgryphiswaldensestrain MSR-1,Magnetospirillummagneticumstrain AMB-1,MagnetococcusmarinusMC-1, Magneto-ovoid bacterium MO-1,DesulfovibriomagneticusRS-1,CandidatusMagnetomorum sp.HK-1,CandidatusMagnetoglobus multicellularis str.Araruama,CandidatusMagnetobacterium bavaricum,andCandidatusDesulfamplus magnetomortis BW-1)using PHASTER (Supplementary Table S3). Most MTB genomes (11 out of 12 genomes) were found to contain predicted prophage regions, with HK-1 being the only exception. Temperate phage genes have been identif ied in 40%-50% of microbial genomes(Canchaya et al., 2003; Casjens, 2003; Fouts, 2006;Paul, 2008; Touchon et al., 2016), and almost 50% of bacterial genomes contain at least one prophage(Touchon et al., 2016). Our results show that prophages may exist in many genomes of MTB.Through lysogenic conversion or transduction,bacteriophages and archaeal viruses contribute to the horizontal transfer of genetic material among microbial genomes (Touchon et al., 2016, 2017).Previous studies have shown that HGT plays an important role in the evolution of magnetotaxis in bacteria (Rioux et al., 2010; Monteil et al., 2018).Alternatively, being infected by phage may contribute to this process.

3.6 Comparative gene content analysis of three Terasakiella strains using reciprocal best matches

We further perform pangenome analysis of SH-1,PR-1, andT.pusilla. As shown in Fig.3, 1 948 gene families were shared among the three species,representing approximately 50% of the proteins in each strain. A total of 428 families were shared by SH-1 and PR-1, but notT.pusilla. The larger overlap between SH-1 and PR-1 suggests that they are more closely related to each other than toT.pusilla. These are mainly involved in signal transduction mechanisms(29.80%), inorganic ion transport and metabolism(23.46%), intracellular traffi cking, secretion and vesicular transport (18.18%) and cell motility(17.05%) (according to COG automatic classif ication of Microscope). These genes may also be associated with magnetosome biosynthesis.

Interestingly, genes related to iron metabolism have been found in the genome ofT.pusillabut not in SH-1 and PR-1 genomes. These genes includedfecR(Anti-FecI sigma factor),fbpC(Fe3+ion import ATPbinding protein) andbfr(bacterioferritin, iron storage and detoxif ication protein). Furthermore, we found 41 copies of histidine kinase in the genome ofT.pusilla,fewer than that in SH-1 (74) and PR-1 (70). However,more copies of methyl-accepting chemotaxis proteins(MCPs) were found inT.pusilla(69) than in SH-1(59) and PR-1 (57), consistent with previous report that high numbers of MCPs are not a common feature of MTB (Ji et al., 2014).

4 CONCLUSION

Comparative genomic analysis of SH-1 performed in the present study revealed that genes involved in glycolysis, the TCA cycle, the non-oxidative phase of the reductive pentose phosphate pathway, PRPP biosynthesis, the ethylmalonyl pathway, and the synthesis of all 20 essential amino acids were identif ied. A fragment of gene encoding haloalkane dehalogenase was also identif ied. ThesoxBAZYXgene cluster,soxCDgenes, andcysCHIgenes were present, indicating that SH-1 is capable of assimilatory sulfate reduction to H2S, and using thiosulfate as electron donor and oxidizing it to sulfate. SH-1 also containednapAB,nasAandnasB, suggesting that it can gain energy by converting nitrate to ammonia.Two predicted prophage regions were found in SH-1,and our results suggest that prophage may be common in MTB. Pangenome analysis showed that diff erences betweenT.pusillaand both SH-1 and PR-1 may be related to signal transduction mechanisms, inorganic ion transport and metabolism, intracellular traffi cking,secretion, vesicular transport and cell motility.

5 DATA AVAILABILITY STATEMENT

All data generated and analyzed during the current study are available from the corresponding author upon request.

6 ACKNOWLEDGMENT

We thank Jianhong XU from Institute of Oceanology, Chinese Academy of Sciences and Weijia ZHANG from Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, for assistance in sampling.