Metagenomic analysis reveals genetic coupling between TonB-dependent transporters and extracellular enzymes in coastal bacterial communities

Abstract

Marine heterotrophic prokaryotes initially release extracellular enzymes to cleave large organic molecules and then take up ambient substrates via transporters. Given the direct influence of extracellular enzymes on nutrient availability, understanding their diversity and dynamics is crucial in comprehending microbial interactions and organic matter cycling in aquatic ecosystems. In this study, metagenomics was employed to investigate the functional diversity and dynamics of extracellular enzymes and transporters in coastal waters over a 22-day period. The metagenome-derived gene pool of organic matter-degrading secretory enzymes and transporters was primarily contributed by three major bacterial classes. Bacteroidota were the primary contributors to the gene pool of secretory carbohydrate-active enzymes (CAZymes), whereas Gammaproteobacteria contribute more to secretory peptidases and TonB-dependent transporters (TBDTs), and Alphaproteobacteria to ATP-binding cassette (ABC) transporters. The distinct substrate targets of the enzymes and transporters combined with the unique dynamics of these taxa across depth layers suggest that organic matter degradation and uptake machinery played a role in ecological niche partitioning. At the community level, the abundance of TBDT genes was more positively correlated with extracellular enzymes than ABC transporters. To further explore taxon-specific differences, we reconstructed 163 bacterial and archaeal metagenome-assembled genomes (MAGs). Correlation patterns at the MAG level varied across taxa: Bacteroidota MAGs exhibited significant positive correlations between TBDTs and extracellular enzymes, whereas Gammaproteobacteria and Alphaproteobacteria MAGs showed weak or no significant correlations. These results suggest the diversity of ecological strategies among marine heterotrophic bacteria and highlight a potential coregulation or functional linkage between extracellular enzymes and TBDTs in the metabolism of marine heterotrophic prokaryotes. Our study advances the understanding of the microbial adaptations driving carbon and nutrient cycling.

Special Topic: Ecology & Environmental Biology.

The online version contains supplementary material available at https://doi.org/10.1007/s42995-025-00314-9.

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Keywords

Bacteria / CAZymes / Peptidases / Transporters / Organic matter assimilation

References

[1] AndersonKL, SalyersAA. Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes. J Bacteriol, 1989, 171: 3192-3198.

[2] ArnostiC. Microbial extracellular enzymes and the marine carbon cycle. Annu Rev Mar Sci, 2011, 3: 401-425.

[3] AzamF, MalfattiF. Microbial structuring of marine ecosystems. Nat Rev Microbiol, 2007, 5: 782-791.

[4] BeidlerI, RobbCS, Vidal-MelgosaS, ZühlkeMK, BartosikD, SolankiV, MarkertS, BecherD, SchwederT, HehemannJH. Marine bacteroidetes use a conserved enzymatic cascade to digest diatom β-mannan. ISME J, 2023, 17: 276-285.

[5] BlanvillainS, MeyerD, BoulangerA, LautierM, GuynetC, DenancéN, VasseJ, LauberE, ArlatM. Plant carbohydrate scavenging through TonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria. PLoS ONE, 2007, 2. e224

[6] BolgerAM, LohseM, UsadelB. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014, 30: 2114-2120.

[7] BowersRM, KyrpidesNC, StepanauskasR, Harmon-SmithM, DoudD, ReddyTBK, SchulzF, JarettJ, RiversAR, Eloe-FadroshEA, TringeSG, IvanovaNN, CopelandA, ClumA, BecraftED, MalmstromRR, BirrenB, PodarM, BorkP, WeinstockGM, et al.. Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea. Nat Biotechnol, 2017, 35: 725-731.

[8] BoydPW, ClaustreH, LevyM, SiegelA, WeberT. Multi-faceted particle pumps drive carbon sequestration in the ocean. Nature, 2019, 568: 327-335.

[9] BuchfinkB, XieC, HusonDH. Fast and sensitive protein alignment using DIAMOND. Nat Methods, 2015, 12: 59-60.

[10] ChaumeilP-A, MussigAJ, HugenholtzP, ParksDH. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics, 2019, 36: 1925-1927.

[11] ChklovskiA, ParksDH, WoodcroftBJ, TysonGW. CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning. Nat Methods, 2023, 20: 1203-1212.

[12] ChrostR, VelimirovB. Measurement of enzyme kinetics in water samples: Effect of freezing and soluble stabilizer. Mar Ecol Prog Ser, 2013, 70: 93-100.

[13] CliffordEL, VarelaMM, De CorteD, BodeA, OrtizV, HerndlGJ, SintesE. Taurine is a major carbon and energy source for marine prokaryotes in the North Atlantic Ocean off the Iberian Peninsula. Microb Ecol, 2019, 78: 299-312.

[14] CrespoBG, PommierT, Fernández-GómezB, Pedrós-AlióC. Taxonomic composition of the particle-attached and free-living bacterial assemblages in the Northwest Mediterranean Sea analyzed by pyrosequencing of the 16S rRNA. MicrobiologyOpen, 2013, 2: 541-552.

[15] DavidsonAL, ChenJ. ATP-binding cassette transporters in bacteria. Annu Rev Biochem, 2004, 73: 241-268.

[16] DavidsonAL, DassaE, OrelleC, ChenJ. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev, 2008, 72: 317-364.

[17] DebeljakP, BayerB, SunY, HerndlGJ, ObernostererI. Seasonal patterns in microbial carbon and iron transporter expression in the Southern Ocean. Microbiome, 2023, 11187.

[18] DrulaE, GarronM-L, DoganS, LombardV, HenrissatB, TerraponN. The carbohydrate-active enzyme database: functions and literature. Nucleic Acids Res, 2021, 50: D571-D577.

[19] FrancisTB, BartosikD, SuraT, SichertA, HehemannJ-H, MarkertS, SchwederT, FuchsBM, TeelingH, AmannRI, BecherD. Changing expression patterns of TonB-dependent transporters suggest shifts in polysaccharide consumption over the course of a spring phytoplankton bloom. ISME J, 2021, 15: 2336-2350.

[20] FuL, NiuB, ZhuZ, WuS, LiW. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics, 2012, 28: 3150-3152.

[21] Gómez-SantosN, GlatterT, KoebnikR, Świątek-PołatyńskaWA, Søgaard-AndersenL. A TonB-dependent transporter is required for secretion of protease PopC across the bacterial outer membrane. Nat Commun, 2019, 101360.

[22] GreenNW, PerdueEM, AikenGR, ButlerKD, ChenH, DittmarT, NiggemannJ, StubbinsA. An intercomparison of three methods for the large-scale isolation of oceanic dissolved organic matter. Mar Chem, 2014, 161: 14-19.

[23] HanY, ZhangM, ChenX, ZhaiW, TanE, TangK. Transcriptomic evidences for microbial carbon and nitrogen cycles in the deoxygenated seawaters of Bohai Sea. Environ Int, 2022, 158. 106889

[24] HopkinsonBM, BarbeauKA. Iron transporters in marine prokaryotic genomes and metagenomes. Environ Microbiol, 2012, 14: 114-128.

[25] HyattD, ChenG-L, LocascioPF, LandML, LarimerFW, HauserLJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinf, 2010, 11119.

[26] JiaoN, LuoT, ChenQ, ZhaoZ, XiaoX, LiuJ, JianZ, XieS, ThomasH, HerndlGJ, BennerR, GonsiorM, ChenF, CaiW-J, RobinsonC. The microbial carbon pump and climate change. Nat Rev Microbiol, 2024, 22: 408-419.

[27] KanehisaM, SatoY, KawashimaM, FurumichiM, TanabeM. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res, 2016, 44: D457-462.

[28] KangDD, LiF, KirtonE, ThomasA, EganR, AnH, WangZ. MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. PeerJ, 2019, 7. e7359

[29] LangmeadB, SalzbergSL. Fast gapped-read alignment with Bowtie 2. Nat Methods, 2012, 9: 357-359.

[30] LiD, LuoR, LiuC-M, LeungC-M, TingH-F, SadakaneK, YamashitaH, LamT-W. MEGAHIT v1.0: A fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods, 2016, 102: 3-11.

[31] Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup. The sequence alignment/map format and SAMtools. Bioinformatics, 2009, 25: 2078-2079.

[32] LiuY, MakarovaKS, HuangW-C, WolfYI, NikolskayaAN, ZhangX, CaiM, ZhangC-J, XuW, LuoZ, ChengL, KooninEV, LiM. Expanded diversity of Asgard archaea and their relationships with eukaryotes. Nature, 2021, 593: 553-557.

[33] LiuL, ChenX, YeJ, MaX, HanY, HeY, TangK. Sulfoquinovose is a widespread organosulfur substrate for Roseobacter clade bacteria in the ocean. ISME J, 2023, 17: 393-405.

[34] LiuS, ChenQ, LiuL, DongC, QiuX, TangK. Organic matter composition fluctuations disrupt free-living bacterial communities more than particle-associated bacterial communities in coastal waters. Sci Total Environ, 2024, 949. 174845

[35] MillerTR, DelcherAL, SalzbergSL, SaundersE, DetterJC, HaldenRU. Genome sequence of the dioxin-mineralizing bacterium Sphingomonas wittichii RW1. J Bacteriol, 2010, 192: 6101-6102.

[36] MoeckGS, CoultonJW. TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport. Mol Microbiol, 1998, 28: 675-681.

[37] MouX, SunS, EdwardsRA, HodsonRE, MoranMA. Bacterial carbon processing by generalist species in the coastal ocean. Nature, 2008, 451: 708-711.

[38] OlmMR, BrownCT, BrooksB, BanfieldJF. dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication. ISME J, 2017, 11: 2864-2868.

[39] PoretskyRS, SunS, MouX, MoranMA. Transporter genes expressed by coastal bacterioplankton in response to dissolved organic carbon. Environ Microbiol, 2010, 12: 616-627.

[40] PriceMN, DehalPS, ArkinAP. FastTree 2 – approximately maximum-likelihood trees for large alignments. PLoS ONE, 2010, 5. e9490

[41] RawlingsND, BarrettAJ, ThomasPD, HuangX, BatemanA, FinnRD. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res, 2017, 46: D624-D632.

[42] ReintjesG, ArnostiC, FuchsBM, AmannR. An alternative polysaccharide uptake mechanism of marine bacteria. ISME J, 2017, 11: 1640-1650.

[43] ReintjesG, ArnostiC, FuchsB, AmannR. Selfish, sharing and scavenging bacteria in the Atlantic Ocean: a biogeographical study of bacterial substrate utilisation. ISME J, 2019, 13: 1119-1132.

[44] RinkeC, ChuvochinaM, MussigAJ, ChaumeilP-A, DavínAA, WaiteDW, WhitmanWB, ParksDH, HugenholtzP. A standardized archaeal taxonomy for the Genome Taxonomy Database. Nat Microbiol, 2021, 6: 946-959.

[45] SaierMH, ReddyVS, Moreno-HagelsiebG, HendargoKJ, ZhangY, IddamsettyV, Lam Katie JingK, TianN, RussumS, WangJ, Medrano-SotoA. The Transporter Classification Database (TCDB): 2021 update. Nucleic Acids Res, 2021, 49: D461-D467.

[46] SchauerK, GougetB, CarrièreM, LabigneA, De ReuseH. Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery. Mol Microbiol, 2007, 63: 1054-1068.

[47] SchauerK, RodionovDA, De ReuseH. New substrates for TonB-dependent transport: do we only see the ‘tip of the iceberg’?. Trends Biochem Sci, 2008, 33: 330-338.

[48] SchlitzerR. Interactive analysis and visualization of geoscience data with Ocean Data View. Comput Geosci, 2002, 28: 1211-1218.

[49] ShultisDD, PurdyMD, BanchsCN, WienerMC. Outer membrane active transport: structure of the BtuB:TonB complex. Science, 2006, 312: 1396-1399.

[50] SieberCMK, ProbstAJ, SharrarA, ThomasBC, HessM, TringeSG, BanfieldJF. Recovery of genomes from metagenomes via a dereplication, aggregation and scoring strategy. Nat Microbiol, 2018, 3: 836-843.

[51] SinsabaughRL, LauberCL, WeintraubMN, AhmedB, AllisonSD, CrenshawC, ContostaAR, CusackD, FreyS, GalloME, GartnerTB, HobbieSE, HollandK, KeelerBL, PowersJS, StursovaM, Takacs-VesbachC, WaldropMP, WallensteinMD, ZakDR, et al.. Stoichiometry of soil enzyme activity at global scale. Ecol Lett, 2008, 11: 1252-1264.

[52] SowellSM, WilhelmLJ, NorbeckAD, LiptonMS, NicoraCD, BarofskyDF, CarlsonCA, SmithRD, GiovanonniSJ. Transport functions dominate the SAR11 metaproteome at low-nutrient extremes in the Sargasso Sea. ISME J, 2009, 3: 93-105.

[53] TangK, JiaoN, LiuK, ZhangY, LiS. Distribution and functions of TonB-dependent transporters in marine bacteria and environments: implications for dissolved organic matter utilization. PLoS ONE, 2012, 7. e41204

[54] TeufelF, Almagro ArmenterosJJ, JohansenAR, JohansenAR, GíslasonMH, PihlSI, TsirigosKD, WintherO, BrunakS, von HeijneG, NielsenH. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol, 2022, 40: 1023-1025.

[55] WagnerGP, KinK, LynchVJ. Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci, 2012, 131: 281-285.

[56] WangF-Q, BartosikD, SidhuC, SiebersR, LuD-C, Trautwein-SchultA, BecherD, HuettelB, RickJ, KirsteinIV, WiltshireKH, SchwederT, FuchsBM, BengtssonMM, TeelingH, AmannRI. Particle-attached bacteria act as gatekeepers in the decomposition of complex phytoplankton polysaccharides. Microbiome, 2024, 1232.

[57] WangX, ArifeenMZU, HouS, ZhengQ. Depth-dependent microbial metagenomes sampled in the northeastern Indian Ocean. Sci Data, 2024, 1188.

[58] XieJ, ChenY, CaiG, CaiR, HuZ, WangH. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res, 2023, 51: W587-W592.

[59] YuanH, LiT, LiH, WangC, LiL, LinX, LinS. Diversity distribution, driving factors and assembly mechanisms of free-living and particle-associated bacterial communities at a Subtropical Marginal Sea. Microorganisms, 2021, 92445.

[60] Zhang J (2015) Ecological continuum from the Changjiang (Yangtze River) watersheds to the East China Sea continental margin. Springer Cham, Switzerland

[61] ZhaoZ, BaltarF, HerndlGJ. Linking extracellular enzymes to phylogeny indicates a predominantly particle-associated lifestyle of deep-sea prokaryotes. Sci Adv, 2020, 6eaaz4354.

[62] ZhaoZ, AmanoC, ReinthalerT, OrellanaMV, HerndlGJ. Substrate uptake patterns shape niche separation in marine prokaryotic microbiome. Sci Adv, 2024, 10eadn5143.

RIGHTS & PERMISSIONS

Ocean University of China