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Abstract
Marine bacterial and archaeal communities control global biogeochemical cycles through nutrient acquisition processes that are ultimately dictated by the metabolic requirements of individual cells. Currently lacking, however, is a sensitive, quick, and quantitative measurement of activity in these single cells. We tested the applicability of copper (I)-catalyzed cycloaddition, or “click”, chemistry to observe and estimate single-cell protein synthesis activity in natural assemblages and isolates of heterotrophic marine bacteria. Incorporation rates of the non-canonical methionine bioortholog L-homopropargylglycine (HPG) were quantified within individual cells by measuring fluorescence of alkyne-conjugated Alexa Fluor® 488 using epifluorescence microscopy. The method’s high sensitivity, along with a conversion factor derived from two Alteromonas spp. Isolates, revealed a broad range of cell-specific protein synthesis within natural microbial populations. Comparison with 35S-methionine microautoradiography showed that a large fraction of the natural marine bacterial assemblage (15-100%), previously considered inactive by autoradiography, were actively synthesizing protein. Data pooled from a large number of samples showed that cell-specific activity scaled logarithmically with cell volume. Assemblage activity distributions of each sample were fit to power-law functions, providing an illustrative and quantitative comparison of assemblages that demonstrate individual protein synthesis rates were commonly partitioned between cells in low- and high-metabolic states in our samples. The HPG method offers a simple potential approach to link individual cell physiology to the ecology and biogeochemistry of bacterial (micro)environments in the ocean.
Highlights
The metabolism and growth responses of marine bacteria and archaea communities significantly affect global ocean ecology and biogeochemical cycles
We found that the high sensitivity and low signal background of the HPG method allowed us to detect a broad spectrum of individual cell activity within natural marine assemblages
FTF bacterial transfer efficiency ranged 69–200% because some cells remained stuck to the filter and transferred cells aggregated in condensation pools on the coverslip
Summary
The metabolism and growth responses of marine bacteria and archaea communities significantly affect global ocean ecology and biogeochemical cycles. Characterizing the adaptive responses, growth, of individual bacteria and archaea (collectively here called “bacteria”) may assist in quantifying the microenvironmental regulation of microbial communities and constrain estimates of their biogeochemical effects. A quantitative individual cell approach should help test the significance of microscale heterogeneity for the maintenance of bacterial genomic and functional diversity in the ocean. Individual cell growth is studied using a variety of approaches. This includes 3Hbased microautoradiography (Fuhrman and Azam, 1982; Cottrell and Kirchman, 2004) and the use of a non-radioisotopic, fluorescence-based method detecting incorporation of the thymidine analog bromodeoxyuridine (BrdU) (Pernthaler et al, 2002; Hamasaki et al, 2004). Other studies have documented mRNA fluorescent in situ hybridization (mRNA FISH; Pernthaler and Amann, 2004) or single-cell analyses of activity using nano secondary ion mass spectrometry (nanoSIMS) to quantify metabolic www.frontiersin.org
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