LETTER

REPLY TO ZHOU AND LI: Plasticity of the genomic haplotype of LETTER elongatus leads to rapid adaptation under laboratory conditions Justin Ungerera, Kristen E. Wendta, John I. Hendryb, Costas D. Maranasb, and Himadri B. Pakrasia,1

Zhou and Li (1) describe a classic phenomenon in mi- Considering the sequencing results reported by crobiology in which the genotypes of rapidly Zhou and Li (1), we find their report and that of Lou evolve to optimize growth under selective conditions. et al. (3) to be consistent with our original work (4). In the original paper describing the fast-growing cya- Zhou and Li report that the Synechococcus 2973 hap- nobacterium Synechococcus elongatus UTEX 2973, lotype (obtained from UTEX) lacks the atpA SNP, Yu et al. (2) described the sequence that de- whereas the premise of Lou et al.’s. report is that Syn- fines the strain. Since 2015, several colleagues who echococcus 7942 with only the atpA SNP grows at the obtained the strain directly from the original Pakrasi same rate as the Synechococcus 2973 strain. In our laboratory stock successfully replicated the 2-h dou- work, we show that Synechococcus 2973 with the bling time of the strain. Seemingly, specific loci affect- atpA SNP removed does grow at the same rate as ing growth rate and light tolerance rapidly interconvert Synechococcus 7942 with only the atpA SNP in- between alternative haplotypes based on the growth cluded (figure 1 of ref. 4). Sequencing results by Zhou conditions. This is confirmed by the sequencing results and Li show that Synechococcus 2973 in their labora- of Zhou and Li (1) who report that the sample in their tory has reverted the atpA SNP; thus, as our data laboratory has mutated toward the Synechococcus show, it grows at the same rate as Synechococcus 7942 haplotype via SNP conversion. In fact, this is sim- 7942-C252Y. ilar to the HL-1 strain of Synechococcus 7942, which Both groups (1, 3) argue that their strains grow they describe as a mutant strain. similarly to Synechococcus 2973; however, neither re- Both Zhou and Li (1) and Lou et al. (3) report that port specific growth rates nor compare their data Synechococcus 7942-C252Y grows similarly to the against a positive control (i.e., haplotype with all three sample of Synechococcus 2973 obtained from UTEX. relevant SNPs). The discrepancy reported by Zhou and However, neither group reports specific growth rates Li (1) clearly arises from the fact that neither they nor or doubling times for direct comparison. Furthermore, Lou et al. (3) seem to have obtained a haplotype iden- Lou et al. (3) reported that the atpA SNP imparts only tical to the “original” Synechococcus 2973. Based on light tolerance similar to that of Synechococcus 2973, the sequencing results, both groups are working with not growth rate. In the course of our work (4), we reg- intermediate haplotypes with intermediate growth ularly verify the complement of SNPs in both wild types rates. We are confident that if Zhou and Li (1) had and mutants thereof. Moreover, we make reciprocal Synechococcus 2973 with the full complement of mutations that demonstrate that removal of individual SNPs, they would find that it grows faster than the SNPs decreases the growth rate of Synechococcus Synechococcus 2973 currently available in their labo- 2973, while adding any individual SNP increases the ratory and the fast Synechococcus 7942-C252Y from growth rate of Synechococcus 7942 (4). Lou et al. (3).

1 Zhou J, Li Y (2018) SNPs deciding the rapid growth of are alterable. Proc Natl Acad Sci USA, 10.1073/pnas.1900210116.

2 Yu J, et al. (2015) Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2. Sci Rep 5:8132.

aDepartment of Biology, Washington University in St. Louis, St. Louis, MO 63130; and bDepartment of Chemical Engineering, Pennsylvania State University, University Park, PA 16802 Author contributions: J.U., K.E.W., J.I.H., C.D.M., and H.B.P. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. 1To whom correspondence should be addressed. Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1900792116 PNAS Latest Articles | 1of2 3 Lou W, et al. (2018) A specific single nucleotide polymorphism in the ATP synthase gene significantly improves environmental stress tolerance of Synechococcus elongatus PCC 7942. Appl Environ Microbiol 84:e01222–e01218. 4 Ungerer J, Wendt KE, Hendry JI, Maranas CD, Pakrasi HB (2018) Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus UTEX 2973. Proc Natl Acad Sci USA 115:E11761–E11770.

2of2 | www.pnas.org/cgi/doi/10.1073/pnas.1900792116 Ungerer et al. LETTER

SNPs deciding the rapid growth of cyanobacteria are alterable LETTER Jie Zhoua and Yin Lia,1

Cyanobacteria researchers are keen to understand cyanobacteria strains were grown under a high light

why Synechococcus elongatus UTEX 2973 (Synecho- intensity of 500 μE or 800 μE, at 38 °C, with 5% CO2. coccus 2973) can grow more than two times faster Two independent experiments and sequencing were than Synechococcus elongatus PCC 7942 (Synecho- performed. coccus 7942), as their genome sequences share 99.8% To our great surprise, no SNP was found in the identity (1). atpA gene, in either S. 2973-1 or S. 7942 HL-1. E260D A recent paper in PNAS by Ungerer et al. (2) gives in Ppnk and Q121R/E134K in RpaA were found in S. an answer. The authors investigate the molecular de- 2973-1, which is consistent with what Ungerer et al. (2) terminants accounting for the rapid growth of Syne- describe for Synechococcus 2973, while Q121R in chococcus 2973. They believe that the rapid growth of RpaA was found in S. 7942 HL-1. This SNP is described Synechococcus 2973 is decided by four SNPs in three in Synechococcus 2973 (2), but not in Synechococcus proteins, namely C252Y in AtpA, E260D in Ppnk, and 7942 HL (3). Q121R/E134K in RpaA. In our laboratory, S. 7942 HL-1 grows as fast as S. In a separate study, Lou et al. (3) coincidently 2973-1 under a high light intensity of 500 μEor

obtained a Synechococcus 7942 mutant (designated 800 μE, at 38 °C, with 5% CO2. However, the SNPs here as Synechococcus 7942 HL), which can grow detected were inconsistent with the published data equally fast compared with that of Synechococcus (2, 3). Our results suggest that the SNPs, which 2973, irrespective of light intensity. Lou et al. indi- were thought to determine the rapid growth of cyano- cated that a single SNP in AtpA (C252Y) accounts for bacteria, are alterable. the increased growth rate of Synechococcus 7942 HL. Thus, we argue that the relationship between Is one mutation (C252Y in AtpA) sufficient to create SNPs and the rapid growth requires further investi- a cyanobacterium mutant growing as fast as Synecho- gation. Perhaps an integrated analysis incorporat- coccus 2973? With this question, we sequenced ing SNPs with data from the genome-scale fluxome (according to ref. 2) the genes atpA, ppnK, and rpaA (4) and transcriptome (5) of Synechococcus 2973 would in Synechococcus 2973 and Synechococcus 7942 HL help gain further insight into the molecular mechanism stored in our laboratory (designated S. 2973-1 and underpinning the rapid growth of Synechococcus 2973. S. 7942 HL-1, respectively). Synechococcus 2973 was Acknowledgments purchased from the Culture Collection of Algae at This work was supported by the Key Research Program of the the University of Texas at Austin, and Synechococcus Chinese Academy of Sciences (ZDRW-ZS-2016-3 to Y.L. and 7942 HL was gift from Xuefeng Lu, Chinese J.Z.), and the National Natural Science Foundation of China Academy of Sciences, Qingdao, China (3). Both (31670048 to J.Z.).

1 Yu J, et al. (2015) Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2. Sci Rep 5:8132. 2 Ungerer J, Wendt KE, Hendry JI, Maranas CD, Pakrasi HB (2018) Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus UTEX 2973. Proc Natl Acad Sci USA 115:E11761–E11770. 3 Lou W, et al. (2018) A specific single nucleotide polymorphism in the ATP synthase gene significantly improves environmental stress tolerance of Synechococcus elongatus PCC 7942. Appl Environ Microbiol 84:e01222–e01218. 4 Hendry JI, et al. (2018) Genome-scale fluxome of Synechococcus elongatus UTEX 2973 using transient 13C-labeling data. Plant Physiol 01357.2018. 5 Tan X, et al. (2018) The primary transcriptome of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Biotechnol Biofuels 11:218.

aChinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Author contributions: J.Z. and Y.L. designed research; J.Z. performed research; J.Z. and Y.L. analyzed data; and J.Z. and Y.L. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. 1To whom correspondence should be addressed. Email: [email protected].

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