Marine bacterioplankton represent a diverse assembly of species differing largely in their abundance, physiology, metabolic activity, and role in microbial food webs. To analyze their sensitivity to bottom-up and top-down controls, we performed a manipulation experiment where grazers were removed, with or without the addition of phosphate. Using amplicon-reads normalization by internal standard (ARNIS), we reconstructed growth curves for almost 300 individual phylotypes. Grazer removal caused a rapid growth of most bacterial groups, which grew at rates of 0.6 to 3.5 day-1, with the highest rates (>4 day-1) recorded among Rhodobacteraceae, Oceanospirillales, Alteromonadaceae, and Arcobacteraceae. Based on their growth response, the phylotypes were divided into three basic groups. Most of the phylotypes responded positively to both grazer removal as well as phosphate addition. The second group (containing, e.g., Rhodobacterales and Rhizobiales) responded to the grazer removal but not to the phosphate addition. Finally, some clades, such as SAR11 and Flavobacteriaceae, responded only to phosphate amendment but not to grazer removal. Our results show large differences in bacterial responses to experimental manipulations at the phylotype level and document different life strategies of marine bacterioplankton. In addition, growth curves of 130 phylogroups of aerobic anoxygenic phototrophs were reconstructed based on changes of the functional pufM gene. The use of functional genes together with rRNA genes may significantly expand the scientific potential of the ARNIS technique. IMPORTANCE Growth is one of the main manifestations of life. It is assumed generally that bacterial growth is constrained mostly by nutrient availability (bottom-up control) and grazing (top-down control). Since marine bacteria represent a very diverse assembly of species with different metabolic properties, their growth characteristics also largely differ accordingly. Currently, the growth of marine microorganisms is typically evaluated using microscopy in combination with fluorescence in situ hybridization (FISH). However, these laborious techniques are limited in their throughput and taxonomical resolution. Therefore, we combined a classical manipulation experiment with next-generation sequencing to resolve the growth dynamics of almost 300 bacterial phylogroups in the coastal Adriatic Sea. The analysis documented that most of the phylogroups responded positively to both grazer removal and phosphate addition. We observed significant differences in growth kinetics among closely related species, which could not be distinguished by the classical FISH technique.

Center Algatech Institute of Microbiology of the Czech Academy of Sciences Třeboň Czechia

Laboratory of Marine Microbiology Institute of Oceanography and Fisheriesgrid 425052 4 Split Croatia

National Marine Fisheries Research Institute Gdynia Poland

Zobrazit více v PubMed

Kirchman DL. 2016. Growth rates of microbes in the oceans. Ann Rev Mar Sci 8:285–309. doi:10.1146/annurev-marine-122414-033938. PubMed DOI

Smith DC, Azam F. 1992. A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3H-leucine. Mar Microb Food Webs 6:107–114.

Kirchman DL. 2001. Measuring bacterial biomass production and growth rates from leucine incorporation in natural aquatic environments. Methods Microbiol 30:227–237. doi:10.1016/S0580-9517(01)30047-8. DOI

Popendorf KJ, Koblížek M, Van Mooy BAS. 2020. Phospholipid turnover rates suggest that bacterial community growth rates in the open ocean are systematically underestimated. Limnol Oceanogr 65:1876–1890. doi:10.1002/lno.11424. DOI

Wright RT, Coffin RB. 1984. Measuring microzooplankton grazing on planktonic marine bacteria by its impact on bacterial production. Microb Ecol 10:137–149. doi:10.1007/BF02011421. PubMed DOI

Rassoulzadegan F, Sheldon RW. 1986. Predator-prey interactions of nanozooplankton and bacteria in an oligotrophic marine environment. Limnol Oceanogr 31:1010–1021. doi:10.4319/lo.1986.31.5.1010. DOI

Silva L, Calleja ML, Huete-Stauffer TM, Ivetic S, Ansari MI, Viegas M, Morán XAG. 2018. Low abundances but high growth rates of coastal heterotrophic bacteria in the Red Sea. Front Microbiol 9:3244. doi:10.3389/fmicb.2018.03244. PubMed DOI PMC

Piwosz K, Shabarova T, Pernthaler J, Posch T, Šimek K, Porcal P, Salcher MM. 2020. Bacterial and eukaryotic small-subunit amplicon data do not provide a quantitative picture of microbial communities, but they are reliable in the context of ecological interpretations. mSphere 5:e00052-20. doi:10.1128/mSphere.00052-20. PubMed DOI PMC

Piwosz K, Shabarova T, Tomasch J, Šimek K, Kopejtka K, Kahl S, Pieper DH, Koblížek M. 2018. Determining lineage-specific bacterial growth curves with a novel approach based on amplicon reads normalization using internal standard (ARNIS). ISME J 12:2640–2654. doi:10.1038/s41396-018-0213-y. PubMed DOI PMC

Koblížek M. 2015. Ecology of aerobic anoxygenic phototrophs in aquatic environments. FEMS Microbiol Rev 39:854–870. doi:10.1093/femsre/fuv032. PubMed DOI

Ferrera I, Gasol JM, Sebastian M, Hojerová E, Koblížek M. 2011. Comparison of growth rates of aerobic anoxygenic phototrophic bacteria and other bacterioplankton groups in coastal Mediterranean waters. Appl Environ Microbiol 77:7451–7458. doi:10.1128/AEM.00208-11. PubMed DOI PMC

Ferrera I, Sánchez O, Kolářová E, Koblížek M, Gasol JM. 2017. Light enhances the growth rates of natural populations of aerobic anoxygenic phototrophic bacteria. ISME J 11:2391–2393. doi:10.1038/ismej.2017.79. PubMed DOI PMC

Eilers H, Pernthaler J, Amann R. 2000. Succession of pelagic marine bacteria during enrichment: a close look at cultivation-induced shifts. Appl Environ Microbiol 66:4634–4640. doi:10.1128/AEM.66.11.4634-4640.2000. PubMed DOI PMC

Yokokawa T, Nagata T, Cottrell MT, Kirchman DL. 2004. Growth rate of the major phylogenetic bacterial groups in the Delaware estuary. Limnol Oceanogr 49:1620–1629. doi:10.4319/lo.2004.49.5.1620. DOI

Teira E, Martínez‐García S, Lønborg C, Álvarez‐Salgado XA. 2009. Growth rates of different phylogenetic bacterioplankton groups in a coastal upwelling system. Environ Microbiol Rep 1:545–554. doi:10.1111/j.1758-2229.2009.00079.x. PubMed DOI

Nikrad MP, Cottrell MT, Kirchman DL. 2014. Growth activity of gammaproteobacterial subgroups in waters off the west Antarctic Peninsula in summer and fall. Environ Microbiol 16:1513–1523. doi:10.1111/1462-2920.12258. PubMed DOI

Sánchez O, Koblížek M, Gasol JM, Ferrera I. 2017. Effects of grazing, phosphorus and light on the growth rates of major bacterioplankton taxa in the coastal NW Mediterranean. Environ Microbiol Rep 9:300–309. doi:10.1111/1758-2229.12535. PubMed DOI

Teira E, Logares R, Gutiérrez‐Barral A, Ferrera I, Varela MM, Morán XAG, Gasol JM. 2019. Impact of grazing, resource availability and light on prokaryotic growth and diversity in the oligotrophic surface global ocean. Environ Microbiol 21:1482–1496. doi:10.1111/1462-2920.14581. PubMed DOI

Sánchez O, Ferrera I, Mabrito I, Gazulla CR, Sebastián M, Auladell A, Marín-Vindas C, Clara Cardelús C, Sanz-Sáez I, Pernice MC, Marrasé C, Sala MM, Gasol JM. 2020. Seasonal impact of grazing, viral mortality, resource availability and light on the group-specific growth rates of coastal Mediterranean bacterioplankton. Sci Rep 10:19773. doi:10.1038/s41598-020-76590-5. PubMed DOI PMC

Long AM, Hou S, Ignacio-Espinoza JC, Fuhrman JA. 2021. Benchmarking microbial growth rate predictions from metagenomes. ISME J 15:183–195. doi:10.1038/s41396-020-00773-1. PubMed DOI PMC

MacArthur HH. 1962. Some generalized theorems of natural selection. Proc Natl Acad Sci USA 48:1893–1897. doi:10.1073/pnas.48.11.1893. PubMed DOI PMC

MacArthur RH, Wilson EO. 1967. The theory of island biogeography, p 203. Princetown Univ Press, Princetown, NJ.

Pianka ER. 1970. On r- and K-selection. Am Nat 104:592–597. doi:10.1086/282697. DOI

Beardsley C, Pernthaler J, Wosniok W, Amann R. 2003. Are readily culturable bacteria in coastal North Sea waters suppressed by selective grazing mortality? Appl Environ Microbiol 69:2624–2630. doi:10.1128/AEM.69.5.2624-2630.2003. PubMed DOI PMC

Allers E, Gómez-Consarnau L, Pinhassi J, Gasol JM, Simek K, Pernthaler J. 2007. Response of Alteromonadaceae and Rhodobacteriaceae to glucose and phosphorus manipulation in marine mesocosms. Environ Microbiol 9:2417–2429. doi:10.1111/j.1462-2920.2007.01360.x. PubMed DOI

Våge S, Storesund JE, Giske J, Thingstad TF. 2014. Optimal defense strategies in an idealized microbial food web under trade-off between competition and defense. PLoS One 9:e101415. doi:10.1371/journal.pone.0101415. PubMed DOI PMC

Giovannoni SJ, Tripp HJ, Givan S, Podar M, Vergin KL, Baptista D, Bibbs L, Eads J, Richardson TH, Noordewier M, Rappé MS, Short JM, Carrington JC, Mathur EJ. 2005. Genome streamlining in a cosmopolitan oceanic bacterium. Science 309:1242–1245. doi:10.1126/science.1114057. PubMed DOI

Yilmaz P, Yarza P, Rapp JZ, Glöckner FO. 2015. Expanding the world of marine bacterial and archaeal clades. Front Microbiol 6:1524. doi:10.3389/fmicb.2015.01524. PubMed DOI PMC

Ngugi DK, Stingl U. 2018. High-quality draft single-cell genome sequence of the NS5 marine group from the Coastal Red Sea. Genome Announc 6:e00565-18. doi:10.1128/genomeA.00565-18. PubMed DOI PMC

Cottrell MT, Kirchman DL. 2000. Natural assemblages of marine proteobacteria and members of the Cytophaga-Flavobacteria cluster consuming low- and high-molecular weight dissolved organic matter. Appl Environ Microbiol 66:1692–1697. doi:10.1128/AEM.66.4.1692-1697.2000. PubMed DOI PMC

Yokokawa T, Nagata T. 2005. Growth and grazing mortality rates of phylogenetic groups of bacterioplankton in coastal marine environments. Appl Environ Microbiol 71:6799–6807. doi:10.1128/AEM.71.11.6799-6807.2005. PubMed DOI PMC

Zheng Q, Lu J, Wang Y, Jiao N. 2019. Genomic reconstructions and potential metabolic strategies of generalist and specialist heterotrophic bacteria associated with an estuary Synechococcus culture. FEMS Microbiol Ecol 95:fiz017. doi:10.1093/femsec/fiz017. PubMed DOI

Sebastián M, Gasol J. 2013. Heterogeneity in the nutrient limitation of different bacterioplankton groups in the Eastern Mediterranean Sea. ISME J 7:1665–1668. doi:10.1038/ismej.2013.42. PubMed DOI PMC

Newton RJ, Griffin LE, Bowles KM, Meile C, Gifford S, Givens CE, Howard EC, King E, Oakley CA, Reisch CR, Rinta-Kanto JM, Sharma S, Sun S, Varaljay V, Vila-Costa M, Westrich JR, Moran MA. 2010. Genome characteristics of a generalist marine bacterial lineage. ISME J 4:784–798. doi:10.1038/ismej.2009.150. PubMed DOI

Yutin N, Suzuki MT, Teeling H, Weber M, Venter JC, Rusch DB, Béjà O. 2007. Assessing diversity and biogeography of aerobic anoxygenic phototrophic bacteria in surface waters of the Atlantic and Pacific Oceans using the Global Ocean Sampling expedition metagenomes. Environ Microbiol 9:1464–1475. doi:10.1111/j.1462-2920.2007.01265.x. PubMed DOI

Waidner LA, Kirchman DL. 2008. Diversity and distribution of ecotypes of the aerobic anoxygenic phototrophy gene pufM in the Delaware Estuary. Appl Environ Microbiol 74:4012–4021. doi:10.1128/AEM.02324-07. PubMed DOI PMC

Ferrera I, Borrego CM, Salazar G, Gasol JM. 2014. Marked seasonality of aerobic anoxygenic phototrophic bacteria in the coastal NW Mediterranean Sea as revealed by cell abundance, pigment concentration and pyrosequencing of pufM gene. Environ Microbiol 16:2953–2965. doi:10.1111/1462-2920.12278. PubMed DOI

Weinbauer MG, Suominen S, Jezbera J, Kerros ME, Marro S, Dolan JR, Šimek K. 2019. Shifts in cell size and community composition of bacterioplankton due to grazing by heterotrophic flagellates: evidence from a marine system. Aquat Microb Ecol 83:295–308. doi:10.3354/ame01919. DOI

Gasol JM. 1994. A framework for the assessment of top-down vs bottom-up control of heterotrophic nanoflagellate abundance. Mar Ecol Prog Ser 113:291–300. doi:10.3354/meps113291. DOI

Gasol JM, Pedrós-Alió C, Vaqué D. 2002. Regulation of bacterial assemblages in oligotrophic plankton systems: results from experimental and empirical approaches. Antonie Van Leeuwenhoek 81:435–452. doi:10.1023/A:1020578418898. PubMed DOI

Solic M, Krstulovic N, Vilibic I, Bojanic N, Kuspipilic G, Sestanovic S, Santic D, Ordulj M. 2009. Variability in the bottom-up and top-down control of bacteria on trophic and temporal scale in the middle Adriatic Sea. Aquat Microb Ecol 58:15–29. doi:10.3354/ame01342. DOI

Solić M, Krstulović N, Kuspilić G, Nincević Gladan Z, Bojanić N, Sestanović S, Santić D, Ordulj M. 2010. Changes in microbial food web structure in response to changed environmental trophic status: a case study of the Vranjic Basin (Adriatic Sea). Mar Environ Res 70:239–249. doi:10.1016/j.marenvres.2010.05.007. PubMed DOI

Šantić D, Piwosz K, Matić F, Vrdoljak Tomaš A, Arapov J, Dean JL, Šolić M, Koblížek M, Kušpilić G, Šestanović S. 2021. Artificial neural network analysis of microbial diversity in the central and southern Adriatic Sea. Sci Rep 11:11186. doi:10.1038/s41598-021-90863-7. PubMed DOI PMC

Han Y, Jiao N, Zhang Y, Zhang F, He C, Liang X, Cai R, Shi Q, Tang K. 2021. Opportunistic bacteria with reduced genomes are effective competitors for organic nitrogen compounds in coastal dinoflagellate blooms. Microbiome 9:71. doi:10.1186/s40168-021-01022-z. PubMed DOI PMC

Chin-Leo G, Kirchman DL. 1990. Unbalanced growth in natural assemblages of marine bacterioplankton. Mar Ecol Prog Ser 63:1–8. doi:10.3354/meps063001. DOI

Sato-Takabe Y, Hamasaki K, Suzuki S. 2019. High temperature accelerates growth of aerobic anoxygenic phototrophic bacteria in seawater. MicrobiologyOpen 8:e00710. doi:10.1002/mbo3.710. PubMed DOI PMC

Koblížek M, Mašín M, Ras J, Poulton AJ, Prášil O. 2007. Rapid growth rates of aerobic anoxygenic phototrophs in the ocean. Environ Microbiol 9:2401–2406. doi:10.1111/j.1462-2920.2007.01354.x. PubMed DOI

Hojerová E, Mašín M, Brunet C, Ferrera I, Gasol JM, Koblížek M. 2011. Distribution and growth of aerobic anoxygenic phototrophs in the Mediterranean Sea. Environ Microbiol 13:2717–2725. doi:10.1111/j.1462-2920.2011.02540.x. PubMed DOI

Orlić M, Dadić V, Grbec B, Leder N, Marki A, Matić F, Mihanović H, Paklar GB, Pasarić M, Pasarić Z, Vilibić I. 2006. Wintertime buoyancy forcing, changing seawater properties and two different circulation systems produced in the Adriatic. J Geophys Res 111:C03S07. doi:10.1029/2005JC003271. DOI

Kasalický V, Zeng Y, Piwosz K, Šimek K, Kratochvilová H, Koblížek M. 2018. Aerobic anoxygenic photosynthesis is commonly present within the genus Limnohabitans. Appl Environ Microbiol 84:e02116-17. doi:10.1128/AEM.02116-17. PubMed DOI PMC

Nercessian O, Noyes E, Kalyuzhnaya MG, Lidstrom ME, Chistoserdova L. 2005. Bacterial populations active in metabolism of C-1 compounds in the sediment of Lake Washington, a freshwater lake. Appl Environ Microbiol 71:6885–6899. doi:10.1128/AEM.71.11.6885-6899.2005. PubMed DOI PMC

Parada AE, Needham DM, Fuhrman JA. 2016. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol 18:1403–1414. doi:10.1111/1462-2920.13023. PubMed DOI

Andrews S. 2010. FastQC: a quality control tool for high throughput sequence data [online]. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.

Martin M. 2011. Cutadapt removes adapter sequences from high throughput sequencing reads. EMBnet J 17:10–12. doi:10.14806/ej.17.1.200. DOI

Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. 2016. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. doi:10.1038/nmeth.3869. PubMed DOI PMC

McMurdie PJ, Holmes S. 2013. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One 8:e61217. doi:10.1371/journal.pone.0061217. PubMed DOI PMC

Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glöckner FO. 2007. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35:7188–7196. doi:10.1093/nar/gkm864. PubMed DOI PMC

Wright ES. 2016. Using DECIPHER v2.0 to analyze big biological sequence data in R. The R J 8:352–359. doi:10.32614/RJ-2016-025. DOI

Béjà O, Suzuki MT, Heidelberg JF, Nelson WC, Preston CM, Hamada T, Eisen JA, Fraser CM, DeLong EF. 2002. Unsuspected diversity among marine aerobic anoxygenic phototrophs. Nature 415:630–633. doi:10.1038/415630a. PubMed DOI

Yutin N, Suzuki MT, Beja O. 2005. Novel primers reveal wider diversity among marine aerobic anoxygenic phototrophs. Appl Environ Microbiol 71:8958–8962. doi:10.1128/AEM.71.12.8958-8962.2005. PubMed DOI PMC

Piwosz K, Vrdoljak A, Frenken T, González-Olalla JM, Šantić D, McKay RM, Spilling K, Guttman L, Znachor P, Mujakić I, Fecskeová LK, Zoccarato L, Hanusová M, Pessina A, Reich T, Grossart HP, Koblížek M. 2020. Light and primary production shape bacterial activity and community composition of aerobic anoxygenic phototrophic bacteria in a microcosm experiment. mSphere 5:e00354-20. doi:10.1128/mSphere.00673-20. PubMed DOI PMC

Wickham H. 2016. ggplot2: elegant graphics for data analysis, Springer-Verlag New York, New York, NY. https://ggplot2.tidyverse.org.

Morris RM, Rappé MS, Connon SA, Vergin KL, Siebold WA, Carlson CA, Giovannoni SJ. 2002. SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420:806–810. doi:10.1038/nature01240. PubMed DOI

Eilers H, Pernthaler J, Peplies J, Glöckner FO, Gerdts G, Amann R. 2001. Isolation of novel pelagic bacteria from the German bight and their seasonal contribution to surface picoplankton. Appl Environ Microbiol 67:5134–5142. doi:10.1128/AEM.67.11.5134-5142.2001. PubMed DOI PMC

Wendeberg A, Pernthaler J, Amann R. 2004. Sensitive multi-color fluorescence in situ hybridization for the identification of environmental microorganisms. Molecular Microb Ecol Manual 3:711–726. doi:10.1007/978-1-4020-2177-0_311. DOI

Marie D, Brussaard C, Partensky F, Vaulot D. 1999. Flow cytometric analysis of phytoplankton, bacteria and viruses. Curr Protoc Cytom Chapter 11:Unit 11.11. doi:10.1002/0471142956.cy1111s10. PubMed DOI

Marie D, Partensky F, Jacquet S, Vaulot D. 1997. Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl Environ Microbiol 63:186–193. doi:10.1128/aem.63.1.186-193.1997. PubMed DOI PMC

Gasol JM, Morán XAG. 2015. Flow cytometric determination of microbial abundances and its use to obtain indices of community structure and relative activity, pp 159–187. In McGenity TJ, Timmis KN, Nogales B. (ed), Hydrocarbon and lipid microbiology protocols. Springer protocols handbooks. Springer, Berlin, Heidelberg, Germany.

Christaki U, Courties C, Massana R, Catala P, Lebaron P, Gasol JM, Zubkov MV. 2011. Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I. Limnol Oceanogr Methods 9:329–339. doi:10.4319/lom.2011.9.329. DOI

Masín M, Zdun A, Ston-Egiert J, Nausch M, Labrenz M, Moulisová V, Koblízek M. 2006. Seasonal changes and diversity of aerobic anoxygenic phototrophs in the Baltic Sea. Aquat Microb Ecol 45:247–254. doi:10.3354/ame045247. DOI

Response of aerobic anoxygenic phototrophic bacteria to limitation and availability of organic carbon

Ecology of aerobic anoxygenic phototrophs on a fine-scale taxonomic resolution in Adriatic Sea unravelled by unsupervised neural network

Growth and mortality of aerobic anoxygenic phototrophs in the North Pacific Subtropical Gyre

Phenology and ecological role of aerobic anoxygenic phototrophs in freshwaters