In this study, we investigated the role for ancestral functional variation that may be selected upon to generate protein functional shifts using ancestral protein resurrection, statistical tests for positive selection, forward and reverse evolutionary genetics, and enzyme functional assays. Data are presented for three instances of protein functional change in the salicylic acid/benzoic acid/theobromine (SABATH) lineage of plant secondary metabolite-producing enzymes. In each case, we demonstrate that ancestral nonpreferred activities were improved upon in a daughter enzyme after gene duplication, and that these functional shifts were likely coincident with positive selection. Both forward and reverse mutagenesis studies validate the impact of one or a few sites toward increasing activity with ancestrally nonpreferred substrates. In one case, we document the occurrence of an evolutionary reversal of an active site residue that reversed enzyme properties. Furthermore, these studies show that functionally important amino acid replacements result in substrate discrimination as reflected in evolutionary changes in the specificity constant (k cat /K M ) for competing substrates, even though adaptive substitutions may affect K M and k cat separately. In total, these results indicate that nonpreferred, or even latent, ancestral protein activities may be coopted at later times to become the primary or preferred protein activities.carboxyl methyltransferase | adaptive protein evolution

An analysis of the microbial metabolism is fundamental to understanding globally important element transformations. One culture-independent approach to deduce those prokaryotic metabolic functions is to analyze metatranscriptomes. Unfortunately, since mRNA is extremely labile, it is unclear whether the abundance patterns detected in nature are vulnerable to considerable modification in situ simply due to sampling procedures. Exemplified on comparisons of metatranscriptomes retrieved from pelagic suboxic zones of the central Baltic Sea (70-120 m depth), earlier identified as areas of high aerobic ammonium oxidation activity, and quantification of specific transcripts in them, we show that different sampling techniques significantly influence the relative abundance of transcripts presumably diagnostic of the habitat. In situ fixation using our newly developed automatic flow injection sampler resulted in an abundance of thaumarchaeal ammonia monooxygenase transcripts that was up to 30-fold higher than that detected in samples obtained using standard oceanographic sampling systems. By contrast, the abundance of transcripts indicative of cellular stress was significantly greater in non-fixed samples. Thus, the importance of in situ fixation in the reliable evaluation of distinct microbial activities in the ecosystem based on metatranscriptomics is obvious. In consequence, our data indicate that the significance of thaumarchaeota to aerobic ammonium oxidation could yet have been considerably underestimated. Taken these results, this could in general also be the case in attempts aimed at an unbiased gene expression analysis of areas below the epipelagic zone, which cover 90% of the world's oceans. The ISME Journal (2012) 6, 461-470; doi:10.1038/ismej.2011.94; published online 21 July 2011 Subject Category: microbial ecology and functional diversity of natural habitats