Click‐chemistry tagging of proteins in living cells: new possibilities for microbial (meta) proteomics

On page 2568 of this issue, Hatzenpichler and colleagues describe the application of BONCAT (bioorthogonal noncanonical amino acid tagging) (Hinz et al., 2013), a clickchemistry method originally developed to study protein synthesis and localization in neuronal cells, to pureculture and environmental bacteria and archaea. Click chemistry and other bioorthogonal reactions have been intensively studied by chemists and some biologists for the past 15 years but have been little used as yet by environmental microbiologists. The authors show that click-chemistry amino acid analogues can be taken up by and detected in a range of pure-culture and environmental bacteria and archaea; that cells identified as translationally active by BONCAT are generally also metabolically active by the independent criterion of ammonia incorporation; and that production of proteins induced by an environmental change (heat shock) can be followed over time. BONCAT and other click-chemistry methods offer a promising route towards minimally invasive, cultivation-free investigations of the in situ enzymatic capabilities of microbes in diverse communities. ‘Bioorthogonal’ describes reactions that occur independently of cellular enzymes and do not affect non-target biomolecules (see Patterson et al., 2014 for a recent overview). In BONCAT and related methods, alkyne–azide additions are used (Fig. 1). Samples are incubated with an amino acid analogue bearing an azide or alkyne group, which is taken up from the medium and incorporated into growing polypeptide chains – ideally without significant toxicity or growth effects. Some analogues (such as the L-azidohomoalanine used here) are recognized by wildtype transfer RNA synthetases, albeit with reduced efficiency, while others can only be incorporated by specially engineered target species (reviewed in Ngo and Tirrell 2011). The azide (or alkyne) group is then a substrate for ring strain-promoted, copper-catalysed or photocatalysed ‘click’ addition of an alkyne(or azide-) containing fluorescent label or capture tag (e.g. biotin), allowing microscopic identification of translationally active microbial cells or the isolation and identification of newly synthesized proteins. In general, the azide group is made part of the internalized compound, as the extreme scarcity of intracellular azides minimizes the risk of background reactions. Biochemical applications of click chemistry reactions have been the subject of several recent reviews (e.g. Baskin and Bertozzi, 2007; Best, 2009; Ostrovskis et al., 2013; Rudolf and Sieber, 2013; Yu and Wang, 2013; Lang and Chin, 2014). Emphasis here will be on the potential for protein-targeted studies of natural microbial communities, but DNA, RNA, lipids and polysaccharides can also be studied by click-chemistry methods (e.g. Jao and Salic, 2008; Raghavan et al., 2008; Dommerholt et al., 2010; Darzynkiewicz et al., 2011; Chang and Bertozzi, 2012; Hagemeijer et al., 2012; Neef et al., 2012). With some of the necessary reagents becoming commercially available, these techniques are increasingly accessible to biologists. One basis of click chemistry is the Staudinger ligation, developed by Bertozzi and co-workers. In their first application, cell-surface azides were produced via an engineered biosynthetic pathway and ligated with biotinylated phosphine, which in turn was detected by fluorescently labelled avidin (Saxon and Bertozzi, 2000). The reaction was soon refined to a ‘traceless’ variant (Fig. 1A), in which the phosphine group is eliminated from the ligation product (Saxon et al., 2000; Saxon and Bertozzi, 2000). While still widely used, the Staudinger ligation is relatively slow, and phosphines are subject to air oxidation. The goal of subsequent work has been to find fast, highly specific reactions that can be carried out in aqueous solutions at physiological temperature and pH, without toxicity to cells or excessive perturbation of the molecules under study, at least over the experimental time course (reviewed in Debets et al., 2013). The target and label molecules must also be able to reach the correct cellular compartment(s), whether by diffusion or uptake. *For correspondence. E-mail bmacgreg@unc.edu; Tel. +1 (919) 260 6038; Fax +1 (919) 962 1254. bs_bs_banner