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Discovery, Taxonomic Distribution, and Phenotypic Characterization of a Gene Required for 3-Methylhopanoid Production

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Discovery, Taxonomic Distribution, and Phenotypic Characterization of a Gene Required for 3-Methylhopanoid Production Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Welander, P. V., and R. E. Summons. “Discovery, Taxonomic Distribution, and Phenotypic Characterization of a Gene Required for 3-methylhopanoid Production.” Proceedings of the National Academy of Sciences 109.32 (2012): 12905–12910. CrossRef. Web. As Published http://dx.doi.org/10.1073/pnas.1208255109 Publisher National Academy of Sciences (U.S.) Version Final published version Citable link http://hdl.handle.net/1721.1/77589 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production Paula V. Welander1 and Roger E. Summons Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, E25-629, Cambridge, MA 02139 Edited by John M. Hayes, Woods Hole Oceanographic Institution, Berkeley, CA, and approved July 2, 2012 (received for review May 15, 2012) Hopanoids methylated at the C-3 position are a subset of bacterial majority of C-3 methylated hopanoid producers are aerobic triterpenoids that are readily preserved in modern and ancient se- methanotrophs. However, the production of 3-methylhopanoids diments and in petroleum. The production of 3-methylhopanoids has also been demonstrated in the acetic acid bacteria (12) indi- by extant aerobic methanotrophs and their common occurrence cating that the taxonomic distribution of 3-methylhopanoids is in modern and fossil methane seep communities, in conjunction not restricted to methanotrophs. Furthermore, recent studies with carbon isotope analysis, has led to their use as biomarker utilizing molecular approaches to identify hopanoid biosynthesis proxies for aerobic methanotrophy. In addition, these lipids are genes in sequenced genomes have highlighted that the diversity of also produced by aerobic acetic acid bacteria and, lacking carbon bacteria capable of producing a specific hopanoid structure could isotope analysis, are more generally used as indicators for aerobio- be underestimated (13–15). These studies have also shown that a sis in ancient ecosystems. However, recent genetic studies have more precise interpretation of hopane hydrocarbon signatures in brought into question our current understanding of the taxonomic both ancient and modern ecosystems requires not only a grasp diversity of methylhopanoid-producing bacteria and have high- of the taxonomic distribution of methylhopanoid producers but lighted that a proper interpretation of methylhopanes in the rock also a deeper understanding of their physiological function in record requires a deeper understanding of their cellular function. In extant bacteria (16, 17). hpnR this study, we identified and deleted a gene, , required for To this end, we employed a combination of microbial genetics, methylation of hopanoids at the C-3 position in the obligate microbial physiology, and bioinformatics analysis to begin to Methylococcus capsulatus methanotroph strain Bath. Bioinfor- understand the biosynthesis and function of C-3 methylated matics analysis revealed that the taxonomic distribution of HpnR hopanoids in Methylococcus capsulatus. A genetic system for extends beyond methanotrophic and acetic acid bacteria. Phenoty- constructing unmarked in-frame deletion mutants was utilized to M. capsulatus hpnR pic analysis of the deletion mutant demon- identify a methylase required for the production of 3-methylho- strated a potential physiological role for 3-methylhopanoids; they panoids. Bioinformatics analysis of this methylase revealed a appear to be required for the maintenance of intracytoplasmic diverse taxonomic distribution beyond the methanotrophic and membranes and cell survival in late stationary phase. Therefore, acetic acid bacteria. Furthermore, phenotypic analysis of the 3-methylhopanoids may prove more useful as proxies for specific C-3 methylase mutant uncovered a potential role for 3-methylho- environmental conditions encountered during stationary phase panoids in late stationary phase survival. These studies highlight rather than a particular bacterial group. the power of combining gene discovery with bioinformatics and physiological analyses to potentially enhance our understanding radical SAM ∣ bacteriohopanepolyols ∣ molecular markers of biomarker signatures in the rock record. opanoids are pentacyclic triterpenoid lipids produced by a Results and Discussion EARTH, ATMOSPHERIC, AND PLANETARY SCIENCES Hvariety of bacteria that are often utilized as geological Identification of a C-3 Methylase in the M. capsulatus Genome. To proxies or biomarkers for certain bacterial species and their identify a protein required for the methylation of hopanoids metabolisms. Among the various hopanoid structures produced at the C-3 position, the genome of M. capsulatus was examined by bacteria (1), those methylated at the C-3 position and those for possible C-3 methylase candidates utilizing search criteria with a penta- and hexafunctionalized amino polar side group based on two previous findings. First, bacterial feeding studies are thought to be primarily produced by Type I and Type X done with labeled methionine have posited that S-adenosyl- methanotrophs (Fig. 1A) (2). As such, the occurrence of these 13 methionine (AdoMet) is a potential methyl donor in the MICROBIOLOGY hopanoids in conjunction with their significant C-depletion in biosynthesis of both 2-methyl and 3-methylhopanoids (12). Sec- modern ecosystems are often utilized as an indicator of metha- notrophic communities (3). In particular, environmental lipid ond, it was recently discovered that a B-12 binding radical analyses have uncovered the existence of aerobic methanotrophy AdoMet protein, HpnP, is required for the production of 2- methylhopanoids in the α-Proteobacterium Rhodopseudomonas in a variety of environments including, for example, the surface palustris sediments of an active marine mud volcano in the Barents Sea (14). Accordingly, we hypothesized that the methylase and in the oxic-anoxic transition zone of the Black Sea water responsible for 3-methylhopanoid production was also a radical column (4, 5). Furthermore, the recalcitrant nature of hopanoid AdoMet protein possibly containing a B-12 binding domain. hydrocarbons allows for their preservation in ancient sediments, which may provide evidence for aerobic metabolisms deep in Author contributions: P.V.W. designed research; P.V.W. performed research; R.E.S. Earth’s history. Although the functionalized amino side group contributed new reagents/analytic tools; P.V.W. and R.E.S. analyzed data; and P.V.W. is lost over time, methylation of the A-ring is retained (6). Thus, and R.E.S. wrote the paper. the presence of C-3 methylated hopanes in sediments 2.5–2.7 bil- The authors declare no conflict of interest. lion years old has been used as one of several lines of molecular This article is a PNAS Direct Submission. and isotopic evidence for Neoarchean aerobiosis (7–11). 1To whom correspondence should be addressed. E-mail: [email protected]. The effectiveness of these specific hopanoids as indicators This article contains supporting information online at www.pnas.org/lookup/suppl/ for aerobic methanotrophy rests partly on the premise that the doi:10.1073/pnas.1208255109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1208255109 PNAS ∣ August 7, 2012 ∣ vol. 109 ∣ no. 32 ∣ 12905–12910 A OH OH OH A OHOH NH2 3 I R R = H aminobacteriohopanepentol (I) II R = CH 3 3-methylaminobacteriohopanepentol (III) OH OH OHOH NH2 3 III IV R Relative Intensity R = H aminobacteriohopanetetrol (II) R = CH 3 3-methylaminobacteriohopanetetrol (IV) B ISMca3 yfiA hyp OrfB 18 20 22 24 26 28 30 32 rpoN hpnR OrfA B 0741 0740 0739 0738 0736 0735 1 kb Fig. 1. Identification of a putative 3-methylhopanoid methylase in M. cap- II I sulatus.(A) The 3-methyl and desmethyl aminobacteriohopanepolyols pro- duced by M. capsulatus. The roman numerals in parenthesis correspond to the structures identified in Fig. 2. (B) Genomic context of the C-3 methylase gene hpnR (MCA0738). Upstream of the gene there is a hypothetical protein (hyp), a putative Sigma-54 modulation protein (yfiA), and the RNA polymer- ase factor Sigma-54 (rpoN). Downstream are two genes that are annotated as ISMca3 transposase genes (OrfA and OrfB). Relative Intensity Using InterPro (http://www.ebi.ac.uk/interpro/), an integrated database of predictive protein signatures, 21 proteins with a M. capsulatus radical AdoMet motif were identified in the gen- 18 20 22 24 26 28 30 32 ome. These 21 proteins were queried against the Acetobacter pas- terurianus genome, an acetic acid bacterium known to produce 3-methylhopanoids (12). Of these 21 proteins, MCA0738 was C the only protein annotated as a B-12 binding radical AdoMet that A. pasterurianus also had a homologue in (e-value of 0). A Basic I Local Alignment Search Tool (BLAST) search of MCA0738 revealed a homologue in other acetic acid bacterial genomes. Although the MCA0738 gene was not surrounded by other II B hopanoid biosynthesis genes on the chromosome (Fig. 1 ), the III occurrence of this particular gene in both M. capsulatus and all sequenced acetic acid bacterial genomes made it an attractive
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