Expression of novel gene content drives adaptation to low iron in the cyanobacterium Acaryochloris

Expression of novel gene content drives adaptation to low iron in the cyanobacterium Acaryochloris Variation in genome content is a potent mechanism of microbial adaptation. The genomes of members of the cyanobacterial genus Acaryochloris vary greatly in gene content as a consequence of the idiosyncratic retention of both recent gene duplicates and plasmid-encoded genes acquired by horizontal transfer. For example, the genome of Acaryochloris strain MBIC11017, which was isolated from an iron-limited environment, is enriched in duplicated and novel genes involved in iron assimilation. Here, we took an integrative approach to characterize the adaptation of Acaryochloris MBIC11017 to low environmental iron availability and the relative contributions of the expression of duplicated versus novel genes. We observed that Acaryochloris MBIC11017 grew faster and to a higher yield in the presence of nanomolar concentrations of iron than did a closely related strain. These differences were associated with both a higher rate of iron assimilation and a greater abun- dance of iron assimilation transcripts. However, recently duplicated genes contributed little to increased transcript dosage; rather, the maintenance of these duplicates in the MBIC11017 genome is likely due to the sharing of ancestral dosage by expression reduction. Instead, novel, horizontally transferred genes are responsible for the differences in transcript abun- dance. The study provides insights on the mechanisms of adaptive genome evolution and gene expression in Acaryochloris. Key words: gene duplication, horizontal gene transfer, expression reduction, positive dosage, adaptation. Introduction populations suggest a key role for gene duplication during The evolution of gene dosage (i.e., gene transcript abun- the adaptation to novel environments (Kondrashov 2012). dance) is an important mechanism of adaptation in pop- This includes adaptation to antibiotics (reviewed by ulations of microorganisms. Gene dosage may be affected Sandegren and Andersson 2009), nutrient limitation by changes in transcriptional regulation or by processes (Cairns and Foster 1991; Brown et al. 1998; Reams and that result in gene copy number variation (CNV) within a Neidle 2003; Gresham et al. 2008), temperature stress genome, including gene duplication (Andersson and (Riehle et al. 2001; James et al. 2008) and heavy metal Hughes 2009; Kondrashov 2012) and horizontal gene exposure (von Rozycki and Nies 2009; Yang et al. 2010; transfer (HGT; Ochman et al. 2000). Although the role of Chow et al. 2012). As a result, the relative contributions of the acquisition of novel genes by HGT (i.e., the flexible both duplication and HGT mechanisms within the same genome) for microbial adaptation to environmental genome during adaptation are not clear. change is well-known (Lawrence and Ochman 1998; Members of the cyanobacterial genus Acaryochloris pro- Ochman et al. 2000; Scho ¨ nknecht et al. 2013), there is vide an excellent system to investigate this issue. The genomes only limited evidence of duplication driving adaptation in of these bacteria, which are unique in their use of the far-red nature (Triglia et al. 1991; Musher et al. 2002; Duraisingh light (>700 nm) absorbing Chlorophyll d (Chl d)as the major and Cowman 2005). This may in part reflect a difficulty of pigment in photosynthesis (Miyashita et al. 1996), have an detection due to the transient nature of many gene dupli- extraordinarily large number of recent gene duplicates com- cates in microorganisms, which may be rapidly deleted pared with other bacterial genomes (Miller et al. 2011). from the genome upon the relaxation of selection during Acaryochloris genomes also contain multiple plasmids laboratory study (Sandegren and Andersson 2009). By (Swingley et al. 2008; unpublished data), and gene content contrast, experimental evolution studies of microbial on these plasmids varies greatly among strains due to their The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1484 Genome Biol. Evol. 10(6):1484–1492. doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE Materials and Methods Culture Conditions All cultures of Acaryochloris strains MBIC11017 and CCMEE 5410 were grown at 30 C with constant shaking at 100 rpm 2 1 and constantillumination of13–18 mmol photons m s cool white fluorescent light. Cultures were either grown in 100 ml media in 250 ml Erlenmeyer flasks or in 600 ml media in 1 l Erlenmeyer flasks. Two types of media were used, one for the high iron condition and one for the low iron condi- tion (Swingley media and Swingley , respectively). Swingley media was prepared as previously described (Swingley et al. 2005, where it was referred to as FeMBG-11). Swingley was prepared as for Swingley media, except for the exclu- sion of ferric ammonium citrate and EDTA iron(III) sodium FIG.1.—The Acaryochloris strain MBIC11017 genome, showing the salt. Ferric iron content in Swingley media is 51 mMand chromosome (outer circle; 6.5 Mbp) and eight plasmids ranging in size 7.7 nM in Swingley . In order to minimize iron contamina- from 121 to 374 kbp (inner circles, 4 scale compared with chromosome; 0 tion, all media was prepared using MilliQ filtered water in a ninth, 2.1 kbp plasmid is not shown). Duplicated and novel iron-assim- ilation genes are color-coded by function, with gene duplicates connected polycarbonate culture flasks that had been soaked overnight by lines. The chromosomal copy of a duplicate pair typically has an ortho- in 1 N HCl. log on the Acaryochloris strain CCMEE 5410 genome and may be consid- ered the parental copy (Miller et al. 2011). Growth Experiments different histories of horizontal acquisition and gene loss Growth was measured as the increase in culture optical den- (Miller et al. 2011). sity at 750 nm (OD ) with a Beckman Coulter DU 530 spec- Although gene duplication in Acaryochloris appears to be trophotometer (Indianapolis, IN). A regression of optical a generally non-adaptive process, with most duplicates density and cell count for both Acaryochloris strains was pro- purged from the genome relatively quickly (Miller et al. duced in order to normalize results to cell count and cell vol- 2011), some retained duplicates are potentially beneficial ume (MBIC11017 cells are smaller than CCMEE 5410, with in their local environments. For example, the genome of approximate diameters of 1.75 and 2.75 mm, respectively). Acaryochloris strain MBIC11017, which was isolated from We performed cell counts with a hemocytometer and took the iron-limited western Pacific Ocean (Miyashita et al. OD readings for a dilution series of cell cultures in mid- 1996), contains multiple duplicated genes that contribute exponential phase. Between 12 and 14 counts were used to different aspects of iron assimilation, including the for each strain. Cell count was then regressed on OD . production of low molecular weight, iron-chelating com- The regressions, which had R values of 0.91 for pounds (siderophores), siderophore transport and transcrip- MBIC11017 and 0.97 for CCMEE 5410, were used to esti- tional regulation (Miller et al. 2011; fig. 1). The MBIC11017 mate cell density from optical density in subsequent genome also contains novel iron assimilation genes that are experiments. not found in the genome of Acaryochloris strain CCMEE 1 8 6 Cells ml ¼ 4  10 ðÞ OD  3  10 ½MBIC 11017 750 5410 (Miller et al. 2011), which was isolated from the iron-replete Salton Sea (Miller et al. 2005). This includes a 1 8 6 Cells ml ¼ 2  10 ðÞ OD þ 3  10 gene cluster involved in siderophore synthesis (Jeanjean ½CCMEE5410 et al. 2008; fig. 1). Here, we show that MBIC11017 exhibits a faster growth For each strain, triplicate independent cultures derived rate and higher cell yield at low iron availability than CCMEE from the same inoculum were grown in Swingley and 5410, as well as a higher rate of iron assimilation. We next Swingley media. Media were prepared as described address whether enhanced fitness of MBIC11017 under low above using 250 ml polycarbonate flasks with a final vol- iron was associated with increased transcript abundance of ume of 100 ml. Approximately 1  10 stationary phase duplicated and/or novel iron assimilation genes. We find that cells from stocks maintained in Swingley medium were novel genes acquired by horizontal transfer are largely respon- used to inoculate each flask. Growth was measured by sible for the differences between strains in iron assimilation taking OD readings every 24–48 h. Generation time gene transcript abundance, whereas duplicated genes are (G) was estimated from the exponential growth phase of principally retained by expression reduction rather than by the culture, as determined by plotting the growth data on positive dosage effects. a semi-log plot, finding time intervals where cultures were Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1485 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE exponentially growing and applying the following 150 ml of a 30% solution of hydrogen peroxide were added formula: to all samples, which were then incubated for 30 min at 60–70 C. Digestions were added to 19 ml of a 2% nitric T  T f 0 G ¼ acid solution for a final acid concentration of approximately OD 3:3 log OD 0 4.4% and a final volume of approximately 20 ml. Optical emission spectroscopy was performed on each sample by T and T are the first and last time points spanning the ex- The University of Montana’s Environmental Biogeochemistry 0 f ponential growth phase. OD and OD are the OD readings Laboratory to determine the amount of iron per milliliter of 0 f 750 corresponding to T and T . culture, which was then normalized to Acaryochloris biomass. 0 f Final yield was estimated by using the final OD reading To account for any iron precipitation, blank controls were from the growth experiment. To determine the effect of strain used. At t , prior to iron addition, no iron was detected in the and condition (i.e., Swingley vs. Swingley media) on final blank control. Iron concentrations in blanked samples after yield, the data were inverse transformed and fit to a linear iron was supplemented were not negligible and varied ap- model. proximately 2-fold. This variation was not meaningful as it is likely the result of accidental aspiration of precipitated iron. Consequently, the iron concentration of all blank samples Iron Addition Experiment were averaged and subtracted from all estimates of iron Approximately 6  10 cells from stocks growing in concentration for samples collected after iron addition Swingley were inoculated into each of five flasks containing (t , t , t ). 12 24 36 600 ml Swingley media and grown as above (supplementary fig. S1, Supplementary Material online). Once the cultures had Cell collection for RNA-seq been in stationary phase for 7 days, cells were harvested for Both strains were grown under three environmental condi- RNA extraction, intracellular iron analysis, and Chl d extrac- tions for RNA-seq analysis (supplementary fig. S2, tion, along with an OD reading (referred to as t ). After 750 0 Supplementary Material online): during exponential growth collecting data for t , culture flasks were supplemented with in low iron media, stationary phase (t of the iron addition EDTA iron(III) sodium salt (28 mM) and ferric ammonium cit- experiment above), and following iron addition (t of the iron rate (23 mM) to attain the iron content of Swingley media addition experiment above). For all conditions, there were five (Swingley et al. 2005). At 12, 24, and 36 h after iron addition independently grown replicate cultures for each strain. For the (t , t , t ), an OD reading was taken and samples were 12 24 36 750 exponential growth condition, both strains were grown to collected for intracellular iron analyses. Cells were also col- mid-exponential phase in Swingley media. 200 ml of cell cul- lected for RNA isolation at t . ture from each sample were collected for RNA isolation. Cell collection was carried out via vacuum filtration onto 1.2 mm pore size polycarbonate membrane filters (MILLIPORE product Intracellular Iron Collection, Digestion, and Analysis RTTP04700). Using sterilized forceps, filters with cells were To determine intracellular iron content, 10 ml of culture from TM carefully inserted into 15 ml Falcon tubes (Corning Inc., each sample was filtered onto 0.6 mm pore size polycarbonate Corning, NY). Tubes containing cells on filters were immedi- membrane filters (MILLIPORE product DTTP02500; ately flashfrozeninliquidnitrogenand stored at 80 Cuntil Burlington, MA). Filters were inserted into 2 ml screw-top RNA extraction. microcentrifuge tubes, and 1 ml of 5 mM EDTA pH 7.8 was added. Tubes were vortexed until cells were resusupended in RNA Extraction the solution, and filters were then removed with a toothpick. Samples were next centrifuged for 10 min at 16,000  gto RNA was isolated by guanidiumthiocyanate-phenol- pellet cells, and the supernatant was aspirated. This process chloroform extraction with PGTX extraction buffer. PGTX was then repeated with 1 ml sterile Swingley media. Cell buffer was prepared as described by Pinto et al. (2009).The pellets were stored at 20 C until chemically digested for extraction protocol used is a combination of the “PGTX 95” iron content analysis using optical emission spectroscopy. protocol outlined in Pinto et al. (2009) and the Qiagen RNeasy TM A modified protocol of EPA method 3050B was used to Mini Handbook with modifications. Falcon tubes contain- digest cell pellets for iron content analysis (Environmental ing cells on polycarbonate filters were removed from the Protection Agency). Millipore water and 70% TraceMetal 80 C freezer and 2 ml warmed PGTX reagent was added. Grade nitric acid (Fisher Scientific, Hampton NH) were added Samples were vortexed to resuspend cells and incubated for in a 1:1 ratio to microcentrifuge tubes containing cell pellets 5min at 95 C with occasional vortexing. Samples were then to a final volume of 1 ml. The tubes were then vortexed to immediately incubated on ice for 5 min following removal of resuspend the pellet and incubated at 85 C for 4 h. Samples filters with sterile pipette tips. Next, 400 ml chloroform was were removed from heat and allowed to cool. Once cool, added, and samples were incubated for 10 min at room 1486 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE temperature with occasional vortexing. Phase separation was differential expression analysis, respectively. Mapped reads then facilitated by centrifugation for 15 min at 4 Cand were assembled using reference GFF annotations. 12,000  g. The aqueous layer was transferred to a new tube, and an equal volume of chloroform was added. Results and Discussion Again, extractions were incubated at room temperature for Acaryochloris MBIC11017 Grows Faster and to a Higher 10 min with occasional vortexing and centrifuged for 15 min Yield than Acaryochloris CCMEE 5410 at Low Iron at 4 C and 12,000  g. To precipitate RNA, the aqueous layer Concentration was transferred to a new tube, 1/10 volume 3 M sodium ac- etate at pH 5.2 and 2.5 volume 100% ice cold ethanol were To test whether MBIC11017 is better adapted to low-iron added. Tubes were mixed by inversion and precipitated over- environments than CCMEE 5410, we assayed growth rate night at 20 C. and cell yield for both strains in media containing either low The following day, samples were briefly chilled to 80 C (7.7 nM) or high (51 mM) concentrations of iron. MBIC11017 and centrifuged 20 min at 4 C and 12,000  g to pellet RNA. grew significantly faster than CCMEE 5410 under both high The supernatant was aspirated, and pellets were washed by and low iron conditions (fig. 2A; F ¼ 203.01, P< 0.0001 (1, 8) resuspension in 1 ml 75% ethanol, and then pelleted again by for the effect of strain in a two-way ANOVA). The difference centrifugation for 10 min at 4 C and 12,000  g. This process in cell size between strains likely contributes to these growth was repeated, and then samples were cleaned according to rate differences: MBIC11017 is smaller than CCMEE 5410 the Qiagen RNeasy Mini RNA Cleanup protocol, including a and thus has a higher surface area to volume ratio, which is DNase step. RNA pellets were resuspended in 100 mlRNase- negatively correlated with bacterial generation time (Banse free water, and 10 ml b-mercaptoethanol was added to 1 mL 1976; Foy 1980). Although strain yields were not statistically buffer RLT to further inhibit RNases. Finally, samples were different under high iron (P ¼ 0.99), MBIC11017 grew to a eluted twice with 35 ml fresh RNase-free water and stored significantly and substantially greater final yield than CCMEE at 80 C. To check for genomic DNA contamination, 25 5410 under low iron (fig. 2B; P< 0.001 by Tukey HSD; rounds of PCR using isiA primers was performed; following F ¼ 19.68 and P ¼ 0.002 for the strain iron interaction). (1, 8) a second DNase treatment, there was no amplification in any We conclude that MBIC11017 is better than CCMEE 5410 of theRNA samples. Samples wereeluted twice with 25 ml at scavenging low concentrations of iron in the environment. fresh RNase-free water. A small aliquot from each sample Further, the statistically similar yields attained by MBIC11017 was taken for quality control and quantification and was under both conditions suggests that its yield was effectively stored at 80 C. limited by a factor other than iron in both treatments. The lower growth rate but similar yield of MBIC11017 at low iron concentration compared with high iron concentration RNA QA/QC, Quantification, Sequencing, and Data may reflect a cost of additional resource allocation toward Analysis iron acquisition in MBIC11017. Fragment analysis of RNA was performed on an Agilent Technologies (Santa Clara, CA) TapeStation using an RNA Acaryochloris MBIC11017 Assimilates Environmental Iron ScreenTape. RIN values for the samples ranged from 6.4 to Faster than Acaryochloris CCMEE 5410 8.2. RNA was quantified using a Qubit2.0 Fluorometer To estimate the rate of iron assimilation from the environment (Thermo Fisher Scientific, Waltham, MA) with the Broad for each strain, we used optical emission spectroscopy to Range RNA Assay Kit. RNA was sent to the Washington monitor the change in intracellular iron content over time. State University, Spokane Genomics Core for library prep Iron was added to stationary phase cultures of both strains with TruSeq Stranded Total with Ribo-zero (Illumina, San to a final concentration of 51 mM Fe (III). Samples were taken Diego, CA) and 50-bp single read sequencing on an immediately prior to (t ) as well as 12, 24, and 36 h after iron Illumina HiSeq-2500. addition (t , t , t ). Prior to iron addition, intracellular iron 12 24 36 Analysis of adapter-trimmed Illumina reads was per- content of both strains was <0.01 fg Fe mm . Following formed using a Galaxy server (Afgan et al. 2016) maintained addition, MBIC11017 assimilated iron three times faster by the University of Montana Genomics Core Laboratory. than CCMEE 5410 (fig. 3;mean6 SE of 0.036 0.006 vs. FASTQC (Andrews 2010) was used to verify sequence qual- 3 1 0.016 0.001 fg Fe mm hr over the first 24 h of the ex- ity. Reads for both Acaryochloris species were mapped to periment; P ¼ 0.0007 for the strain time interaction). their respective genome assemblies (CCMEE 5410: acces- sion GCA_000238775.2; MBIC11017: accession GCA_ Higher Levels of Expression of Iron Assimilation Genes in 000018105.1) using Bowtie2 (Langmead and Salzberg Acaryochloris MBIC11017 2012). The resulting sorted BAM files were analyzed using HT-seq Count (Anders et al. 2015) and DESeq2 (Love et al. Bacterial iron assimilation proteins include siderophores, 2014) to count the number of transcripts and perform siderophore transporters and Fur transcriptional regulators. Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1487 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE FIG.2.—(A) Growth rate in generations per day for Acaryochloris strains under low and high iron conditions. Error bars indicate standard errors. (B)Final yield in cells ml for Acaryochloris strains grown under low and high iron conditions. Error bars indicate standard errors. FIG.3.—Cell volume-normalized intracellular iron content of Acaryochloris strains following iron addition. Error bars indicate standard errors. Siderophores are low molecular weight compounds with a FIG.4.—Heatmap of log -transformed normalized gene counts for high affinity for iron that are transported out of the cell by iron assimilation genes in the transport, Fur homolog, and siderophore ATP-binding cassette (ABC) superfamily proteins (Kranzler categories, respectively. et al. 2013). Siderophores with chelated iron are shuttled back into the cell by TonB-dependent transporters (Kranzler et al. 2013). Transcription of many siderophore synthesis and above); and 3) cells 36 h after iron addition (t ). To compare transporter genes are regulated by Fur transcriptional regula- gene expression between strains, we estimated the log -fold tors. In E. coli and other enteric bacteria, Fur regulators exhibit difference in normalized gene counts (Love et al. 2014). metal-dependent repression (Escolaretal. 1999): when intra- The transcriptomic data were tightly associated with ob- cellular iron is high, transcription of genes under Fur regula- served physiological differences between MBIC11017 and tion is repressed, whereas transcription is derepressed when CCMEE 5410. The abundance of transcripts involved in side- intracellular iron is low (Andrews et al. 2003). rophore production and iron transport was greater in To evaluate whether phenotypic differences between MBIC110017 under all conditions tested (fig. 4), consistent strains are associated with a greater relative abundance of with the higher rate of iron assimilation (fig. 3) and greater yield iron assimilation transcripts in MBIC11017, we performed an at low iron (fig. 2B) of this strain. The expression of Fur homo- RNA-seq analysis (supplementary fig. S2, Supplementary logs was also greater in MBIC11017 for the sw and t treat- 0 0 Material online) for the following conditions: 1) exponential ments (fig. 4). In Gram-negative bacteria, Fur is not only a phase cells growing at low iron concentration (sw ); 2) station- repressor of iron-assimilation genes but is also a major regula- ary phase cells (t from the iron addition experiment described tory protein with diverse functions (Ratledge and Dover 2000). 1488 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE FIG.5.—Heatmap of log -transformed normalized gene expression for Class 1 genes (single-copy orthologs present in both genomes). Cells represent individual replicates. It is therefore difficult to interpret the effects of individual Expression Reduction of Iron Assimilation Duplicates in Acaryochloris fur homologs without characterizing these MBIC11017 genes. Given the toxicity of iron at high concentrations and For Class 2 genes (duplicated in MBIC11017, single-copy in the increased number of iron assimilation genes under regu- CCMEE 5410), we can distinguish between different potential latory control, we do speculate that these genes may be mechanisms of MBIC11017 gene duplicate retention. Greater important for the maintenance of iron homeostasis in aggregate transcript abundance of MBIC11017 duplicates MBIC11017. compared with expression of the CCMEE 5410 copy would What contributes to this greater abundance of iron assim- be consistent with a positive dosage effect (Kondrashov ilation transcripts in MBIC10017? Potential mechanisms dis- 2012). Alternatively, similar expression between duplicates cussed below include the stronger expression of single-copy and the CCMEE 5410 copy would provide evidence that se- orthologs present in both genomes (Class 1 genes), the en- lection to maintain ancestral levels of gene expression follow- hanced expression of duplicated genes in MBIC11017 that ing duplication resulted in expression reduction and the are single-copy in CCMEE 5410 (Class 2 genes) and the ex- retention of duplicates in the MBIC11017 genome (Qian pression of genes that are novel to the MBIC11017 genome et al. 2010). Expression between strains was similar for (Class 3 genes). most loci: only two of the nine Class 2 genes had log -fold differences of at least 2 (fig. 6; supplementary table S2, Supplementary Material online). One of these encodes a Little Expression Divergence between Single-Copy TonB transporter, which had greater expression in CCMEE Orthologs 5410 at t and t . The other encodes a Fur transcriptional 0 36 Five of the 17 Class 1 genes (single-copy in both genomes) regulator which had greater expression in MBIC11017 under had a log -fold difference in expression between strains all treatments. These results imply that expression reduction with an absolute value of at least 2 for at least one condition has played a more important role for the maintenance of (fig. 5; supplementary table S1, Supplementary Material on- MBIC11017 duplicates than has positive dosage. line). Of these, two were more highly expressed in Expression reduction can arise by chance or by selection to MBIC11017. One is an annotated ExbD biopolymer trans- avoid maladaptive stoichiometry following duplication, and it porter gene (AM1_RS00740) with greater expression at t has been reported to be an important mechanism of duplicate and t . The second was an annotated (Fur) transcriptional retentioninmammals andyeast (Qian et al. 2010; Lan and repressor (AM1_RS05030) with greater expression during Pritchard 2016). In human and mouse, most young tandem exponential growth (sw ) and stationary phase (t ). Three duplicates are down-regulated and appear to be retained by 0 0 genes exhibited higher expression in CCMEE 5410: two an- the sharing of ancestral levels of dosage (Lan and Pritchard notated ABC transporters at t (ON05_RS11755, 2016). This may be a result of the epigenetic silencing of ON05_RS03170) and a siderophore-producing gene (ferric duplicates by increased promoter methylation (Rodin and aerobactin receptor ON05_RS11635) at t . We conclude Riggs 2003), which is known to decrease downstream gene that expression divergence of single-copy orthologs does expression in mammals (Weber et al. 2007). Consistent with not appear to make a major contribution to the physiolog- this model, the promoters of young duplicates in human are ical differences between strains. generally heavily methylated (Keller and Yi 2014). Whether a Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1489 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE FIG.6.—Heatmap of log -transformed normalized gene expression for Class 2 genes (duplicated in MBIC11017, single-copy in CCMEE 5410). Cells represent individual replicates (for MBIC11017, expression is summed over paralogs). FIG.7.—Heatmap of log -transformed normalized gene expression for Class 3 genes (no ortholog in CCMEE 5410). Cells represent individual replicates. Chr corresponds to genes located on the MBIC11017 chromosome; P1 and P2 correspond to paralogs located on plasmids. similar mechanism contributes to expression reduction in Transcription of Novel Genes Drives Stronger Expression of Acaryochloris remains to be investigated, but there is some Iron Assimilation Genes in MBIC11017 evidence for the regulation of gene expression by methylation Of the 25 Class 3 genes (no ortholog in CCMEE 5410), three in bacteria (Casadesus and Low 2006; Adhikari and Curtis have also been duplicated and 20 are encoded on plasmids. 2016). Most (>80%) plasmid-encoded genes in Acaryochloris are Gene expression of duplicates was also often asymmetric. In idiosyncratic to either MBIC11017 or CCMEE 5410, which a majority of cases, the ancestral chromosomal copy had a suggests that they have been acquired by HGT (Miller et al. greater estimated transcript abundance across all conditions 2011). This includes a cluster of genes (AM1_RS29740- (supplementary table S3, Supplementary Material online). AM1_RS29790; fig. 1) flanked by transposase genes that is However, there was also limited evidence for condition- homologous to a characterized siderophore-producing gene dependent differences in which copy was more highly cluster in the cyanobacterium Anabaena PCC 7120 (Jeanjean expressed (supplementary table S3, Supplementary Material et al. 2008). This cluster accounts for the vast majority of online). For example, for the transporter gene exbB, one of Acaryochloris transcripts that map to siderophore-producing the plasmid copies was the most highly expressed copy for genes (figs. 4, 5,and 7). Certain Class 3 genes exhibited sw and t , whereas the chromosomal copy was most highly 0 0 particularly high transcript abundance. For example, a expressed at t . This observed expression divergence among plasmid-encoded annotated TonB-dependent siderophore copies may be indicative of either sub-functionalization or neo- receptor (AM1_RS29960) was highly expressed under all functionalization of duplicates (Moore and Purugganan 2005). 1490 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE Afgan E, et al. 2016. The Galaxy platform for accessible, reproducible and conditions, and in the top 20 most highly expressed protein- collaborative biomedical analyses: 2016 update. Nucleic Acids Res. coding genes during stationary phase t (supplementary ta- 44(W1):W3–W10. ble S4, Supplementary Material online). These results indi- Anders S, Pyl PT, Huber W. 2015. HTSeq: a Python framework to work cate that novel HGT-derived gene content in the with high-throughput sequencing data. Bioinformatics MBIC11017 genome accounts for the majority of the differ- 31(2):166–169. Andersson DI, Hughes D. 2009. Gene amplification and adaptive evolution ences in iron assimilation gene dosage between in bacteria. Annu Rev Genet. 43:167–195. Acaryochloris strains. Although the nature of the interac- Andrews S. 2010. FastQC: a quality control tool for high throughput se- tions among these novel and ancestral Acaryochloris iron quence data. Available from: http://www.bioinformatics.babraham. assimilation proteins remains to be investigated, it is of par- ac.uk/projects/fastqc, last accessed June 4, 2018. ticular interest whether the highly expressed siderophore Andrews SC, Robinson AK, Rodr ıguez-Quin ~ones F. 2003. Bacterial iron homeostasis. FEMS Microbiol Rev. 27(2–3):215–237. receptor AM1_RS29960 recognizes iron-chelated sidero- Banse K. 1976. Rates of growth, respiration and photosynthesis of uni- phores produced by AM1_RS29740-AM1_RS29790. cellular algae as related to cell size. A review. J Phycol. 12:135–140. Brown CJ, Todd KM, Rosenzweig RF. 1998. Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited Concluding Remarks environment. Mol Biol Evol. 15(8):931–942. One implication of the observed pattern of expression reduc- Cairns J, Foster PL. 1991. Adaptive reversion of a frameshift mutation in tion of duplicates in MBIC11017 is that there has been selec- Escherichia coli. Genetics 128(4):695–701. tion to maintain proper stoichiometry among the ancestral Casadesus J, Low D. 2006. Epigenetic gene regulation in the bacterial world. Microbiol Mol Biol Rev. 70(3):830–856. components of iron assimilation. According to this scenario, Chow EWL, Morrow CA, Djordjevic JT, Wood IA, Fraser JA. 2012. reductions in duplicate expression occurred before the dele- Microevolution of Cryptococcus neoformans driven by massive tan- tion of one of the copies, thereby favoring their retention. This dem gene amplification. Mol Biol Evol. 29(8):1987–2000. is consistent with the evidence for strong purifying selection Duraisingh MT, Cowman AF. 2005. Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop. 94(3):181–190. (d /d  0.1) on retained duplicates in Acaryochloris (Miller N S Escolar L, Perez-Martın J, De Lorenzo V. 1999. Opening the iron box: et al. 2011). By contrast, we suggest that the activities of transcriptional metalloregulation by the Fur protein. J Bacteriol. the novel, horizontally transferred iron assimilation genes 181(20):6223–6229. that have become established in the Acaryochloris Foy RH. 1980. The influence of surface to volume ratio on the growth rates MBIC11017 genome may be largely independent of ancestral of planktonic blue-green algae. Br Phycol J. 15(3):279–289. iron metabolism. In particular, we might expect this to be the Gresham D, et al. 2008. The repertoire and dynamics of evolutionary adaptations to controlled nutrient-limited environments in yeast. case for the highly expressed siderophore cluster, since genes PLoS Genet. 4(12):e1000303. involved in secondary metabolism (such as siderophore syn- Jain R, Rivera MC, Lake JA. 1999. Horizontal gene transfer among thesis) are more likely to be successfully horizontally trans- genomes: the complexity hypothesis. Proc Natl Acad Sci U S A. ferred than those that encode proteins that participate in 96(7):3801–3806. diverse interactions with the existing proteome (Jain et al. James TC, Usher J, Campbell S, Bond U. 2008. Lager yeasts possess dy- namic genomes that undergo rearrangements and gene amplification 1999; Nakamura et al. 2004; Puigbo et al. 2010). Further in response to stress. Curr Genet. 53(3):139–152. clarification of these issues will ultimately provide a deeper Jeanjean R, et al. 2008. A large gene cluster encoding peptide synthetases understanding of how novel and duplicated genes are incor- and polyketide synthases is involved in production of siderophores and porated into an existing expression network. oxidative stress response in the cyanobacterium Anabaena sp. strain PCC 7120. Environ Microbiol. 10(10):2574–2585. Keller TE, Yi SV. 2014. DNA methylation and evolution of duplicate genes. Supplementary Material Proc Natl Acad Sci U S A. 111(16):5932–5937. Kondrashov FA. 2012. Gene duplication as a mechanism of genomic adap- Supplementary data areavailableat Genome Biology and tation to a changing environment. Proc Biol Sci. 279(1749):5048–5057. Evolution online. Kranzler C, Rudolf M, Keren N, Schleiff E. 2013. Iron in cyanobacteria. Adv Bot Res. 65:57–105. Lan X, Pritchard JK. 2016. Coregulation of tandem duplicate genes slows Acknowledgments evolution of subfunctionalization in mammals. Science 352(6288): We thank Emiko Sano for technical advice and Jim Elser, John 1009–1013. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie McCutcheon, Frank Rosenzweig, and two anonymous 2. Nat Methods 9(4):357–359. reviewers for comments on the manuscript. This work was Lawrence JG, Ochman H. 1998. Molecular archaeology of the Escherichia supported by award NNA15BB04A from the National coli genome. Proc Natl Acad Sci U S A. 95(16):9413–9417. Aeronautics and Space Administration. Love MI, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15(12):550. Literature Cited Miller SR, et al. 2005. Discovery of a free-living chlorophyll d-producing cyanobacterium with a hybrid proteobacterial/cyanobacterial small- Adhikari S, Curtis PD. 2016. DNA methyltransferases and epigenetic reg- subunit rRNA gene. Proc Natl Acad Sci U S A. 102(3):850–855. ulation in bacteria. FEMS Microbiol Rev. 40(5):575–591. Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1491 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE Miller SR, Wood AM, Blankenship RE, Kim M, Ferriera S. 2011. Dynamics Riehle MM, Bennett AF, Long AD. 2001. Genetic architecture of thermal of gene duplication in the genomes of chlorophyll d-producing cya- adaptation in Escherichia coli. Proc Natl Acad Sci U S A. nobacteria: implications for the ecological niche. Genome Biol Evol. 98(2):525–530. 3:601–613. Rodin SN, Riggs AD. 2003. Epigenetic silencing may aid evolution by gene Miyashita H, et al. 1996. Chlorophyll d as a major pigment. Nature duplication. J Mol Evol. 56(6):718–729. 383(6599):402. Sandegren L, Andersson DI. 2009. Bacterial gene amplification: implica- Moore RC, Purugganan MD. 2005. The evolutionary dynamics of plant tions for the evolution of antibiotic resistance. Nat Rev Microbiol. duplicate genes. Curr Opin Plant Biol. 8(2):122–128. 7:578–588. Musher DM, et al. 2002. Emergence of macrolide resistance during treat- Scho ¨ nknecht G, et al. 2013. Gene transfer from bacteria and archaea ment of pneumococcal pneumonia. N Engl J Med. 346(8):630. facilitated evolution of an extremophilic eukaryote. Science Nakamura Y, Itoh T, Matsuda H, Gojobori T. 2004. Biased biological func- 339:1207–1210. tions of horizontally transferred genes in prokaryotic genomes. Nat Swingley WD, Hohmann-Marriott MF, Le Olson T, Blankenship RE. 2005. Genet. 36(7):760–766. Effect of iron on growth and ultrastructure of Acaryochloris marina. Ochman H, Lawrence JG, Groisman EA. 2000. Lateral gene transfer and Appl Environ Microbiol. 71(12):8606–8610. the nature of bacterial innovation. Nature 405(6784):299–304. Swingley WD, et al. 2008. Niche adaptation and genome expansion in the Pinto FL, Thapper A, Sontheim W, Lindblad P. 2009. Analysis of current chlorophyll d-producing cyanobacterium Acaryochloris marina.Proc and alternative phenol based RNA extraction methodologies for cya- Natl Acad Sci U S A. 105:2005–2010. nobacteria. BMC Mol Biol. 10:79. Triglia T, Foote SJ, Kemp DJ, Cowman AF. 1991. Amplification of the Puigbo  P, Wolf YI, Koonin EV. 2010. The tree and net components of multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen prokaryote evolution. Genome Biol Evol. 2:745–756. as multiple independent events. Mol Cell Biol. 11:5244. Qian W, Liao B-Y, Chang AY-F, Zhang J. 2010. Maintenance of duplicate von Rozycki T, Nies DH. 2009. Cupriavidus metallidurans: evolution of a genes and their functional redundancy by reduced expression. Trends metal-resistant bacterium. Antonie Van Leeuwenhoek 96(2): Genet. 26(10):425–430. 115–139. Ratledge C, Dover LG. 2000. Iron metabolism in pathogenic bacteria. Weber M, et al. 2007. Distribution, silencing potential and evolutionary Annu Rev Microbiol. 54:881–941. impact of promoter DNA methylation in the human genome. Nat Reams AB, Neidle EL. 2003. Genome plasticity in Acinetobacter: Genet. 39:457–466. new degradative capabilities acquired by the spontaneous ampli- Yang F, et al. 2010. Biosequestration via cooperative binding of copper by fication of large chromosomal segments. Mol Microbiol. 47(5): Ralstonia pickettii. Environ Technol. 31:1045–1060. 1291–1304. Associate editor: Takashi Gojobori 1492 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genome Biology and Evolution Oxford University Press

Expression of novel gene content drives adaptation to low iron in the cyanobacterium Acaryochloris

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Abstract

Variation in genome content is a potent mechanism of microbial adaptation. The genomes of members of the cyanobacterial genus Acaryochloris vary greatly in gene content as a consequence of the idiosyncratic retention of both recent gene duplicates and plasmid-encoded genes acquired by horizontal transfer. For example, the genome of Acaryochloris strain MBIC11017, which was isolated from an iron-limited environment, is enriched in duplicated and novel genes involved in iron assimilation. Here, we took an integrative approach to characterize the adaptation of Acaryochloris MBIC11017 to low environmental iron availability and the relative contributions of the expression of duplicated versus novel genes. We observed that Acaryochloris MBIC11017 grew faster and to a higher yield in the presence of nanomolar concentrations of iron than did a closely related strain. These differences were associated with both a higher rate of iron assimilation and a greater abun- dance of iron assimilation transcripts. However, recently duplicated genes contributed little to increased transcript dosage; rather, the maintenance of these duplicates in the MBIC11017 genome is likely due to the sharing of ancestral dosage by expression reduction. Instead, novel, horizontally transferred genes are responsible for the differences in transcript abun- dance. The study provides insights on the mechanisms of adaptive genome evolution and gene expression in Acaryochloris. Key words: gene duplication, horizontal gene transfer, expression reduction, positive dosage, adaptation. Introduction populations suggest a key role for gene duplication during The evolution of gene dosage (i.e., gene transcript abun- the adaptation to novel environments (Kondrashov 2012). dance) is an important mechanism of adaptation in pop- This includes adaptation to antibiotics (reviewed by ulations of microorganisms. Gene dosage may be affected Sandegren and Andersson 2009), nutrient limitation by changes in transcriptional regulation or by processes (Cairns and Foster 1991; Brown et al. 1998; Reams and that result in gene copy number variation (CNV) within a Neidle 2003; Gresham et al. 2008), temperature stress genome, including gene duplication (Andersson and (Riehle et al. 2001; James et al. 2008) and heavy metal Hughes 2009; Kondrashov 2012) and horizontal gene exposure (von Rozycki and Nies 2009; Yang et al. 2010; transfer (HGT; Ochman et al. 2000). Although the role of Chow et al. 2012). As a result, the relative contributions of the acquisition of novel genes by HGT (i.e., the flexible both duplication and HGT mechanisms within the same genome) for microbial adaptation to environmental genome during adaptation are not clear. change is well-known (Lawrence and Ochman 1998; Members of the cyanobacterial genus Acaryochloris pro- Ochman et al. 2000; Scho ¨ nknecht et al. 2013), there is vide an excellent system to investigate this issue. The genomes only limited evidence of duplication driving adaptation in of these bacteria, which are unique in their use of the far-red nature (Triglia et al. 1991; Musher et al. 2002; Duraisingh light (>700 nm) absorbing Chlorophyll d (Chl d)as the major and Cowman 2005). This may in part reflect a difficulty of pigment in photosynthesis (Miyashita et al. 1996), have an detection due to the transient nature of many gene dupli- extraordinarily large number of recent gene duplicates com- cates in microorganisms, which may be rapidly deleted pared with other bacterial genomes (Miller et al. 2011). from the genome upon the relaxation of selection during Acaryochloris genomes also contain multiple plasmids laboratory study (Sandegren and Andersson 2009). By (Swingley et al. 2008; unpublished data), and gene content contrast, experimental evolution studies of microbial on these plasmids varies greatly among strains due to their The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1484 Genome Biol. Evol. 10(6):1484–1492. doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE Materials and Methods Culture Conditions All cultures of Acaryochloris strains MBIC11017 and CCMEE 5410 were grown at 30 C with constant shaking at 100 rpm 2 1 and constantillumination of13–18 mmol photons m s cool white fluorescent light. Cultures were either grown in 100 ml media in 250 ml Erlenmeyer flasks or in 600 ml media in 1 l Erlenmeyer flasks. Two types of media were used, one for the high iron condition and one for the low iron condi- tion (Swingley media and Swingley , respectively). Swingley media was prepared as previously described (Swingley et al. 2005, where it was referred to as FeMBG-11). Swingley was prepared as for Swingley media, except for the exclu- sion of ferric ammonium citrate and EDTA iron(III) sodium FIG.1.—The Acaryochloris strain MBIC11017 genome, showing the salt. Ferric iron content in Swingley media is 51 mMand chromosome (outer circle; 6.5 Mbp) and eight plasmids ranging in size 7.7 nM in Swingley . In order to minimize iron contamina- from 121 to 374 kbp (inner circles, 4 scale compared with chromosome; 0 tion, all media was prepared using MilliQ filtered water in a ninth, 2.1 kbp plasmid is not shown). Duplicated and novel iron-assim- ilation genes are color-coded by function, with gene duplicates connected polycarbonate culture flasks that had been soaked overnight by lines. The chromosomal copy of a duplicate pair typically has an ortho- in 1 N HCl. log on the Acaryochloris strain CCMEE 5410 genome and may be consid- ered the parental copy (Miller et al. 2011). Growth Experiments different histories of horizontal acquisition and gene loss Growth was measured as the increase in culture optical den- (Miller et al. 2011). sity at 750 nm (OD ) with a Beckman Coulter DU 530 spec- Although gene duplication in Acaryochloris appears to be trophotometer (Indianapolis, IN). A regression of optical a generally non-adaptive process, with most duplicates density and cell count for both Acaryochloris strains was pro- purged from the genome relatively quickly (Miller et al. duced in order to normalize results to cell count and cell vol- 2011), some retained duplicates are potentially beneficial ume (MBIC11017 cells are smaller than CCMEE 5410, with in their local environments. For example, the genome of approximate diameters of 1.75 and 2.75 mm, respectively). Acaryochloris strain MBIC11017, which was isolated from We performed cell counts with a hemocytometer and took the iron-limited western Pacific Ocean (Miyashita et al. OD readings for a dilution series of cell cultures in mid- 1996), contains multiple duplicated genes that contribute exponential phase. Between 12 and 14 counts were used to different aspects of iron assimilation, including the for each strain. Cell count was then regressed on OD . production of low molecular weight, iron-chelating com- The regressions, which had R values of 0.91 for pounds (siderophores), siderophore transport and transcrip- MBIC11017 and 0.97 for CCMEE 5410, were used to esti- tional regulation (Miller et al. 2011; fig. 1). The MBIC11017 mate cell density from optical density in subsequent genome also contains novel iron assimilation genes that are experiments. not found in the genome of Acaryochloris strain CCMEE 1 8 6 Cells ml ¼ 4  10 ðÞ OD  3  10 ½MBIC 11017 750 5410 (Miller et al. 2011), which was isolated from the iron-replete Salton Sea (Miller et al. 2005). This includes a 1 8 6 Cells ml ¼ 2  10 ðÞ OD þ 3  10 gene cluster involved in siderophore synthesis (Jeanjean ½CCMEE5410 et al. 2008; fig. 1). Here, we show that MBIC11017 exhibits a faster growth For each strain, triplicate independent cultures derived rate and higher cell yield at low iron availability than CCMEE from the same inoculum were grown in Swingley and 5410, as well as a higher rate of iron assimilation. We next Swingley media. Media were prepared as described address whether enhanced fitness of MBIC11017 under low above using 250 ml polycarbonate flasks with a final vol- iron was associated with increased transcript abundance of ume of 100 ml. Approximately 1  10 stationary phase duplicated and/or novel iron assimilation genes. We find that cells from stocks maintained in Swingley medium were novel genes acquired by horizontal transfer are largely respon- used to inoculate each flask. Growth was measured by sible for the differences between strains in iron assimilation taking OD readings every 24–48 h. Generation time gene transcript abundance, whereas duplicated genes are (G) was estimated from the exponential growth phase of principally retained by expression reduction rather than by the culture, as determined by plotting the growth data on positive dosage effects. a semi-log plot, finding time intervals where cultures were Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1485 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE exponentially growing and applying the following 150 ml of a 30% solution of hydrogen peroxide were added formula: to all samples, which were then incubated for 30 min at 60–70 C. Digestions were added to 19 ml of a 2% nitric T  T f 0 G ¼ acid solution for a final acid concentration of approximately OD 3:3 log OD 0 4.4% and a final volume of approximately 20 ml. Optical emission spectroscopy was performed on each sample by T and T are the first and last time points spanning the ex- The University of Montana’s Environmental Biogeochemistry 0 f ponential growth phase. OD and OD are the OD readings Laboratory to determine the amount of iron per milliliter of 0 f 750 corresponding to T and T . culture, which was then normalized to Acaryochloris biomass. 0 f Final yield was estimated by using the final OD reading To account for any iron precipitation, blank controls were from the growth experiment. To determine the effect of strain used. At t , prior to iron addition, no iron was detected in the and condition (i.e., Swingley vs. Swingley media) on final blank control. Iron concentrations in blanked samples after yield, the data were inverse transformed and fit to a linear iron was supplemented were not negligible and varied ap- model. proximately 2-fold. This variation was not meaningful as it is likely the result of accidental aspiration of precipitated iron. Consequently, the iron concentration of all blank samples Iron Addition Experiment were averaged and subtracted from all estimates of iron Approximately 6  10 cells from stocks growing in concentration for samples collected after iron addition Swingley were inoculated into each of five flasks containing (t , t , t ). 12 24 36 600 ml Swingley media and grown as above (supplementary fig. S1, Supplementary Material online). Once the cultures had Cell collection for RNA-seq been in stationary phase for 7 days, cells were harvested for Both strains were grown under three environmental condi- RNA extraction, intracellular iron analysis, and Chl d extrac- tions for RNA-seq analysis (supplementary fig. S2, tion, along with an OD reading (referred to as t ). After 750 0 Supplementary Material online): during exponential growth collecting data for t , culture flasks were supplemented with in low iron media, stationary phase (t of the iron addition EDTA iron(III) sodium salt (28 mM) and ferric ammonium cit- experiment above), and following iron addition (t of the iron rate (23 mM) to attain the iron content of Swingley media addition experiment above). For all conditions, there were five (Swingley et al. 2005). At 12, 24, and 36 h after iron addition independently grown replicate cultures for each strain. For the (t , t , t ), an OD reading was taken and samples were 12 24 36 750 exponential growth condition, both strains were grown to collected for intracellular iron analyses. Cells were also col- mid-exponential phase in Swingley media. 200 ml of cell cul- lected for RNA isolation at t . ture from each sample were collected for RNA isolation. Cell collection was carried out via vacuum filtration onto 1.2 mm pore size polycarbonate membrane filters (MILLIPORE product Intracellular Iron Collection, Digestion, and Analysis RTTP04700). Using sterilized forceps, filters with cells were To determine intracellular iron content, 10 ml of culture from TM carefully inserted into 15 ml Falcon tubes (Corning Inc., each sample was filtered onto 0.6 mm pore size polycarbonate Corning, NY). Tubes containing cells on filters were immedi- membrane filters (MILLIPORE product DTTP02500; ately flashfrozeninliquidnitrogenand stored at 80 Cuntil Burlington, MA). Filters were inserted into 2 ml screw-top RNA extraction. microcentrifuge tubes, and 1 ml of 5 mM EDTA pH 7.8 was added. Tubes were vortexed until cells were resusupended in RNA Extraction the solution, and filters were then removed with a toothpick. Samples were next centrifuged for 10 min at 16,000  gto RNA was isolated by guanidiumthiocyanate-phenol- pellet cells, and the supernatant was aspirated. This process chloroform extraction with PGTX extraction buffer. PGTX was then repeated with 1 ml sterile Swingley media. Cell buffer was prepared as described by Pinto et al. (2009).The pellets were stored at 20 C until chemically digested for extraction protocol used is a combination of the “PGTX 95” iron content analysis using optical emission spectroscopy. protocol outlined in Pinto et al. (2009) and the Qiagen RNeasy TM A modified protocol of EPA method 3050B was used to Mini Handbook with modifications. Falcon tubes contain- digest cell pellets for iron content analysis (Environmental ing cells on polycarbonate filters were removed from the Protection Agency). Millipore water and 70% TraceMetal 80 C freezer and 2 ml warmed PGTX reagent was added. Grade nitric acid (Fisher Scientific, Hampton NH) were added Samples were vortexed to resuspend cells and incubated for in a 1:1 ratio to microcentrifuge tubes containing cell pellets 5min at 95 C with occasional vortexing. Samples were then to a final volume of 1 ml. The tubes were then vortexed to immediately incubated on ice for 5 min following removal of resuspend the pellet and incubated at 85 C for 4 h. Samples filters with sterile pipette tips. Next, 400 ml chloroform was were removed from heat and allowed to cool. Once cool, added, and samples were incubated for 10 min at room 1486 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE temperature with occasional vortexing. Phase separation was differential expression analysis, respectively. Mapped reads then facilitated by centrifugation for 15 min at 4 Cand were assembled using reference GFF annotations. 12,000  g. The aqueous layer was transferred to a new tube, and an equal volume of chloroform was added. Results and Discussion Again, extractions were incubated at room temperature for Acaryochloris MBIC11017 Grows Faster and to a Higher 10 min with occasional vortexing and centrifuged for 15 min Yield than Acaryochloris CCMEE 5410 at Low Iron at 4 C and 12,000  g. To precipitate RNA, the aqueous layer Concentration was transferred to a new tube, 1/10 volume 3 M sodium ac- etate at pH 5.2 and 2.5 volume 100% ice cold ethanol were To test whether MBIC11017 is better adapted to low-iron added. Tubes were mixed by inversion and precipitated over- environments than CCMEE 5410, we assayed growth rate night at 20 C. and cell yield for both strains in media containing either low The following day, samples were briefly chilled to 80 C (7.7 nM) or high (51 mM) concentrations of iron. MBIC11017 and centrifuged 20 min at 4 C and 12,000  g to pellet RNA. grew significantly faster than CCMEE 5410 under both high The supernatant was aspirated, and pellets were washed by and low iron conditions (fig. 2A; F ¼ 203.01, P< 0.0001 (1, 8) resuspension in 1 ml 75% ethanol, and then pelleted again by for the effect of strain in a two-way ANOVA). The difference centrifugation for 10 min at 4 C and 12,000  g. This process in cell size between strains likely contributes to these growth was repeated, and then samples were cleaned according to rate differences: MBIC11017 is smaller than CCMEE 5410 the Qiagen RNeasy Mini RNA Cleanup protocol, including a and thus has a higher surface area to volume ratio, which is DNase step. RNA pellets were resuspended in 100 mlRNase- negatively correlated with bacterial generation time (Banse free water, and 10 ml b-mercaptoethanol was added to 1 mL 1976; Foy 1980). Although strain yields were not statistically buffer RLT to further inhibit RNases. Finally, samples were different under high iron (P ¼ 0.99), MBIC11017 grew to a eluted twice with 35 ml fresh RNase-free water and stored significantly and substantially greater final yield than CCMEE at 80 C. To check for genomic DNA contamination, 25 5410 under low iron (fig. 2B; P< 0.001 by Tukey HSD; rounds of PCR using isiA primers was performed; following F ¼ 19.68 and P ¼ 0.002 for the strain iron interaction). (1, 8) a second DNase treatment, there was no amplification in any We conclude that MBIC11017 is better than CCMEE 5410 of theRNA samples. Samples wereeluted twice with 25 ml at scavenging low concentrations of iron in the environment. fresh RNase-free water. A small aliquot from each sample Further, the statistically similar yields attained by MBIC11017 was taken for quality control and quantification and was under both conditions suggests that its yield was effectively stored at 80 C. limited by a factor other than iron in both treatments. The lower growth rate but similar yield of MBIC11017 at low iron concentration compared with high iron concentration RNA QA/QC, Quantification, Sequencing, and Data may reflect a cost of additional resource allocation toward Analysis iron acquisition in MBIC11017. Fragment analysis of RNA was performed on an Agilent Technologies (Santa Clara, CA) TapeStation using an RNA Acaryochloris MBIC11017 Assimilates Environmental Iron ScreenTape. RIN values for the samples ranged from 6.4 to Faster than Acaryochloris CCMEE 5410 8.2. RNA was quantified using a Qubit2.0 Fluorometer To estimate the rate of iron assimilation from the environment (Thermo Fisher Scientific, Waltham, MA) with the Broad for each strain, we used optical emission spectroscopy to Range RNA Assay Kit. RNA was sent to the Washington monitor the change in intracellular iron content over time. State University, Spokane Genomics Core for library prep Iron was added to stationary phase cultures of both strains with TruSeq Stranded Total with Ribo-zero (Illumina, San to a final concentration of 51 mM Fe (III). Samples were taken Diego, CA) and 50-bp single read sequencing on an immediately prior to (t ) as well as 12, 24, and 36 h after iron Illumina HiSeq-2500. addition (t , t , t ). Prior to iron addition, intracellular iron 12 24 36 Analysis of adapter-trimmed Illumina reads was per- content of both strains was <0.01 fg Fe mm . Following formed using a Galaxy server (Afgan et al. 2016) maintained addition, MBIC11017 assimilated iron three times faster by the University of Montana Genomics Core Laboratory. than CCMEE 5410 (fig. 3;mean6 SE of 0.036 0.006 vs. FASTQC (Andrews 2010) was used to verify sequence qual- 3 1 0.016 0.001 fg Fe mm hr over the first 24 h of the ex- ity. Reads for both Acaryochloris species were mapped to periment; P ¼ 0.0007 for the strain time interaction). their respective genome assemblies (CCMEE 5410: acces- sion GCA_000238775.2; MBIC11017: accession GCA_ Higher Levels of Expression of Iron Assimilation Genes in 000018105.1) using Bowtie2 (Langmead and Salzberg Acaryochloris MBIC11017 2012). The resulting sorted BAM files were analyzed using HT-seq Count (Anders et al. 2015) and DESeq2 (Love et al. Bacterial iron assimilation proteins include siderophores, 2014) to count the number of transcripts and perform siderophore transporters and Fur transcriptional regulators. Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1487 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE FIG.2.—(A) Growth rate in generations per day for Acaryochloris strains under low and high iron conditions. Error bars indicate standard errors. (B)Final yield in cells ml for Acaryochloris strains grown under low and high iron conditions. Error bars indicate standard errors. FIG.3.—Cell volume-normalized intracellular iron content of Acaryochloris strains following iron addition. Error bars indicate standard errors. Siderophores are low molecular weight compounds with a FIG.4.—Heatmap of log -transformed normalized gene counts for high affinity for iron that are transported out of the cell by iron assimilation genes in the transport, Fur homolog, and siderophore ATP-binding cassette (ABC) superfamily proteins (Kranzler categories, respectively. et al. 2013). Siderophores with chelated iron are shuttled back into the cell by TonB-dependent transporters (Kranzler et al. 2013). Transcription of many siderophore synthesis and above); and 3) cells 36 h after iron addition (t ). To compare transporter genes are regulated by Fur transcriptional regula- gene expression between strains, we estimated the log -fold tors. In E. coli and other enteric bacteria, Fur regulators exhibit difference in normalized gene counts (Love et al. 2014). metal-dependent repression (Escolaretal. 1999): when intra- The transcriptomic data were tightly associated with ob- cellular iron is high, transcription of genes under Fur regula- served physiological differences between MBIC11017 and tion is repressed, whereas transcription is derepressed when CCMEE 5410. The abundance of transcripts involved in side- intracellular iron is low (Andrews et al. 2003). rophore production and iron transport was greater in To evaluate whether phenotypic differences between MBIC110017 under all conditions tested (fig. 4), consistent strains are associated with a greater relative abundance of with the higher rate of iron assimilation (fig. 3) and greater yield iron assimilation transcripts in MBIC11017, we performed an at low iron (fig. 2B) of this strain. The expression of Fur homo- RNA-seq analysis (supplementary fig. S2, Supplementary logs was also greater in MBIC11017 for the sw and t treat- 0 0 Material online) for the following conditions: 1) exponential ments (fig. 4). In Gram-negative bacteria, Fur is not only a phase cells growing at low iron concentration (sw ); 2) station- repressor of iron-assimilation genes but is also a major regula- ary phase cells (t from the iron addition experiment described tory protein with diverse functions (Ratledge and Dover 2000). 1488 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE FIG.5.—Heatmap of log -transformed normalized gene expression for Class 1 genes (single-copy orthologs present in both genomes). Cells represent individual replicates. It is therefore difficult to interpret the effects of individual Expression Reduction of Iron Assimilation Duplicates in Acaryochloris fur homologs without characterizing these MBIC11017 genes. Given the toxicity of iron at high concentrations and For Class 2 genes (duplicated in MBIC11017, single-copy in the increased number of iron assimilation genes under regu- CCMEE 5410), we can distinguish between different potential latory control, we do speculate that these genes may be mechanisms of MBIC11017 gene duplicate retention. Greater important for the maintenance of iron homeostasis in aggregate transcript abundance of MBIC11017 duplicates MBIC11017. compared with expression of the CCMEE 5410 copy would What contributes to this greater abundance of iron assim- be consistent with a positive dosage effect (Kondrashov ilation transcripts in MBIC10017? Potential mechanisms dis- 2012). Alternatively, similar expression between duplicates cussed below include the stronger expression of single-copy and the CCMEE 5410 copy would provide evidence that se- orthologs present in both genomes (Class 1 genes), the en- lection to maintain ancestral levels of gene expression follow- hanced expression of duplicated genes in MBIC11017 that ing duplication resulted in expression reduction and the are single-copy in CCMEE 5410 (Class 2 genes) and the ex- retention of duplicates in the MBIC11017 genome (Qian pression of genes that are novel to the MBIC11017 genome et al. 2010). Expression between strains was similar for (Class 3 genes). most loci: only two of the nine Class 2 genes had log -fold differences of at least 2 (fig. 6; supplementary table S2, Supplementary Material online). One of these encodes a Little Expression Divergence between Single-Copy TonB transporter, which had greater expression in CCMEE Orthologs 5410 at t and t . The other encodes a Fur transcriptional 0 36 Five of the 17 Class 1 genes (single-copy in both genomes) regulator which had greater expression in MBIC11017 under had a log -fold difference in expression between strains all treatments. These results imply that expression reduction with an absolute value of at least 2 for at least one condition has played a more important role for the maintenance of (fig. 5; supplementary table S1, Supplementary Material on- MBIC11017 duplicates than has positive dosage. line). Of these, two were more highly expressed in Expression reduction can arise by chance or by selection to MBIC11017. One is an annotated ExbD biopolymer trans- avoid maladaptive stoichiometry following duplication, and it porter gene (AM1_RS00740) with greater expression at t has been reported to be an important mechanism of duplicate and t . The second was an annotated (Fur) transcriptional retentioninmammals andyeast (Qian et al. 2010; Lan and repressor (AM1_RS05030) with greater expression during Pritchard 2016). In human and mouse, most young tandem exponential growth (sw ) and stationary phase (t ). Three duplicates are down-regulated and appear to be retained by 0 0 genes exhibited higher expression in CCMEE 5410: two an- the sharing of ancestral levels of dosage (Lan and Pritchard notated ABC transporters at t (ON05_RS11755, 2016). This may be a result of the epigenetic silencing of ON05_RS03170) and a siderophore-producing gene (ferric duplicates by increased promoter methylation (Rodin and aerobactin receptor ON05_RS11635) at t . We conclude Riggs 2003), which is known to decrease downstream gene that expression divergence of single-copy orthologs does expression in mammals (Weber et al. 2007). Consistent with not appear to make a major contribution to the physiolog- this model, the promoters of young duplicates in human are ical differences between strains. generally heavily methylated (Keller and Yi 2014). Whether a Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1489 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE FIG.6.—Heatmap of log -transformed normalized gene expression for Class 2 genes (duplicated in MBIC11017, single-copy in CCMEE 5410). Cells represent individual replicates (for MBIC11017, expression is summed over paralogs). FIG.7.—Heatmap of log -transformed normalized gene expression for Class 3 genes (no ortholog in CCMEE 5410). Cells represent individual replicates. Chr corresponds to genes located on the MBIC11017 chromosome; P1 and P2 correspond to paralogs located on plasmids. similar mechanism contributes to expression reduction in Transcription of Novel Genes Drives Stronger Expression of Acaryochloris remains to be investigated, but there is some Iron Assimilation Genes in MBIC11017 evidence for the regulation of gene expression by methylation Of the 25 Class 3 genes (no ortholog in CCMEE 5410), three in bacteria (Casadesus and Low 2006; Adhikari and Curtis have also been duplicated and 20 are encoded on plasmids. 2016). Most (>80%) plasmid-encoded genes in Acaryochloris are Gene expression of duplicates was also often asymmetric. In idiosyncratic to either MBIC11017 or CCMEE 5410, which a majority of cases, the ancestral chromosomal copy had a suggests that they have been acquired by HGT (Miller et al. greater estimated transcript abundance across all conditions 2011). This includes a cluster of genes (AM1_RS29740- (supplementary table S3, Supplementary Material online). AM1_RS29790; fig. 1) flanked by transposase genes that is However, there was also limited evidence for condition- homologous to a characterized siderophore-producing gene dependent differences in which copy was more highly cluster in the cyanobacterium Anabaena PCC 7120 (Jeanjean expressed (supplementary table S3, Supplementary Material et al. 2008). This cluster accounts for the vast majority of online). For example, for the transporter gene exbB, one of Acaryochloris transcripts that map to siderophore-producing the plasmid copies was the most highly expressed copy for genes (figs. 4, 5,and 7). Certain Class 3 genes exhibited sw and t , whereas the chromosomal copy was most highly 0 0 particularly high transcript abundance. For example, a expressed at t . This observed expression divergence among plasmid-encoded annotated TonB-dependent siderophore copies may be indicative of either sub-functionalization or neo- receptor (AM1_RS29960) was highly expressed under all functionalization of duplicates (Moore and Purugganan 2005). 1490 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Iron Adaptation in Acaryochloris GBE Afgan E, et al. 2016. The Galaxy platform for accessible, reproducible and conditions, and in the top 20 most highly expressed protein- collaborative biomedical analyses: 2016 update. Nucleic Acids Res. coding genes during stationary phase t (supplementary ta- 44(W1):W3–W10. ble S4, Supplementary Material online). These results indi- Anders S, Pyl PT, Huber W. 2015. HTSeq: a Python framework to work cate that novel HGT-derived gene content in the with high-throughput sequencing data. Bioinformatics MBIC11017 genome accounts for the majority of the differ- 31(2):166–169. Andersson DI, Hughes D. 2009. Gene amplification and adaptive evolution ences in iron assimilation gene dosage between in bacteria. Annu Rev Genet. 43:167–195. Acaryochloris strains. Although the nature of the interac- Andrews S. 2010. FastQC: a quality control tool for high throughput se- tions among these novel and ancestral Acaryochloris iron quence data. Available from: http://www.bioinformatics.babraham. assimilation proteins remains to be investigated, it is of par- ac.uk/projects/fastqc, last accessed June 4, 2018. ticular interest whether the highly expressed siderophore Andrews SC, Robinson AK, Rodr ıguez-Quin ~ones F. 2003. Bacterial iron homeostasis. FEMS Microbiol Rev. 27(2–3):215–237. receptor AM1_RS29960 recognizes iron-chelated sidero- Banse K. 1976. Rates of growth, respiration and photosynthesis of uni- phores produced by AM1_RS29740-AM1_RS29790. cellular algae as related to cell size. A review. J Phycol. 12:135–140. Brown CJ, Todd KM, Rosenzweig RF. 1998. Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited Concluding Remarks environment. Mol Biol Evol. 15(8):931–942. One implication of the observed pattern of expression reduc- Cairns J, Foster PL. 1991. Adaptive reversion of a frameshift mutation in tion of duplicates in MBIC11017 is that there has been selec- Escherichia coli. Genetics 128(4):695–701. tion to maintain proper stoichiometry among the ancestral Casadesus J, Low D. 2006. Epigenetic gene regulation in the bacterial world. Microbiol Mol Biol Rev. 70(3):830–856. components of iron assimilation. According to this scenario, Chow EWL, Morrow CA, Djordjevic JT, Wood IA, Fraser JA. 2012. reductions in duplicate expression occurred before the dele- Microevolution of Cryptococcus neoformans driven by massive tan- tion of one of the copies, thereby favoring their retention. This dem gene amplification. Mol Biol Evol. 29(8):1987–2000. is consistent with the evidence for strong purifying selection Duraisingh MT, Cowman AF. 2005. Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop. 94(3):181–190. (d /d  0.1) on retained duplicates in Acaryochloris (Miller N S Escolar L, Perez-Martın J, De Lorenzo V. 1999. Opening the iron box: et al. 2011). By contrast, we suggest that the activities of transcriptional metalloregulation by the Fur protein. J Bacteriol. the novel, horizontally transferred iron assimilation genes 181(20):6223–6229. that have become established in the Acaryochloris Foy RH. 1980. The influence of surface to volume ratio on the growth rates MBIC11017 genome may be largely independent of ancestral of planktonic blue-green algae. Br Phycol J. 15(3):279–289. iron metabolism. In particular, we might expect this to be the Gresham D, et al. 2008. The repertoire and dynamics of evolutionary adaptations to controlled nutrient-limited environments in yeast. case for the highly expressed siderophore cluster, since genes PLoS Genet. 4(12):e1000303. involved in secondary metabolism (such as siderophore syn- Jain R, Rivera MC, Lake JA. 1999. Horizontal gene transfer among thesis) are more likely to be successfully horizontally trans- genomes: the complexity hypothesis. Proc Natl Acad Sci U S A. ferred than those that encode proteins that participate in 96(7):3801–3806. diverse interactions with the existing proteome (Jain et al. James TC, Usher J, Campbell S, Bond U. 2008. Lager yeasts possess dy- namic genomes that undergo rearrangements and gene amplification 1999; Nakamura et al. 2004; Puigbo et al. 2010). Further in response to stress. Curr Genet. 53(3):139–152. clarification of these issues will ultimately provide a deeper Jeanjean R, et al. 2008. A large gene cluster encoding peptide synthetases understanding of how novel and duplicated genes are incor- and polyketide synthases is involved in production of siderophores and porated into an existing expression network. oxidative stress response in the cyanobacterium Anabaena sp. strain PCC 7120. Environ Microbiol. 10(10):2574–2585. Keller TE, Yi SV. 2014. DNA methylation and evolution of duplicate genes. Supplementary Material Proc Natl Acad Sci U S A. 111(16):5932–5937. Kondrashov FA. 2012. Gene duplication as a mechanism of genomic adap- Supplementary data areavailableat Genome Biology and tation to a changing environment. Proc Biol Sci. 279(1749):5048–5057. Evolution online. Kranzler C, Rudolf M, Keren N, Schleiff E. 2013. Iron in cyanobacteria. Adv Bot Res. 65:57–105. Lan X, Pritchard JK. 2016. Coregulation of tandem duplicate genes slows Acknowledgments evolution of subfunctionalization in mammals. Science 352(6288): We thank Emiko Sano for technical advice and Jim Elser, John 1009–1013. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie McCutcheon, Frank Rosenzweig, and two anonymous 2. Nat Methods 9(4):357–359. reviewers for comments on the manuscript. This work was Lawrence JG, Ochman H. 1998. Molecular archaeology of the Escherichia supported by award NNA15BB04A from the National coli genome. Proc Natl Acad Sci U S A. 95(16):9413–9417. Aeronautics and Space Administration. Love MI, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15(12):550. Literature Cited Miller SR, et al. 2005. Discovery of a free-living chlorophyll d-producing cyanobacterium with a hybrid proteobacterial/cyanobacterial small- Adhikari S, Curtis PD. 2016. DNA methyltransferases and epigenetic reg- subunit rRNA gene. Proc Natl Acad Sci U S A. 102(3):850–855. ulation in bacteria. FEMS Microbiol Rev. 40(5):575–591. Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 1491 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018 Gallagher and Miller GBE Miller SR, Wood AM, Blankenship RE, Kim M, Ferriera S. 2011. Dynamics Riehle MM, Bennett AF, Long AD. 2001. Genetic architecture of thermal of gene duplication in the genomes of chlorophyll d-producing cya- adaptation in Escherichia coli. Proc Natl Acad Sci U S A. nobacteria: implications for the ecological niche. Genome Biol Evol. 98(2):525–530. 3:601–613. Rodin SN, Riggs AD. 2003. Epigenetic silencing may aid evolution by gene Miyashita H, et al. 1996. Chlorophyll d as a major pigment. Nature duplication. J Mol Evol. 56(6):718–729. 383(6599):402. Sandegren L, Andersson DI. 2009. Bacterial gene amplification: implica- Moore RC, Purugganan MD. 2005. The evolutionary dynamics of plant tions for the evolution of antibiotic resistance. Nat Rev Microbiol. duplicate genes. Curr Opin Plant Biol. 8(2):122–128. 7:578–588. Musher DM, et al. 2002. Emergence of macrolide resistance during treat- Scho ¨ nknecht G, et al. 2013. Gene transfer from bacteria and archaea ment of pneumococcal pneumonia. N Engl J Med. 346(8):630. facilitated evolution of an extremophilic eukaryote. Science Nakamura Y, Itoh T, Matsuda H, Gojobori T. 2004. Biased biological func- 339:1207–1210. tions of horizontally transferred genes in prokaryotic genomes. Nat Swingley WD, Hohmann-Marriott MF, Le Olson T, Blankenship RE. 2005. Genet. 36(7):760–766. Effect of iron on growth and ultrastructure of Acaryochloris marina. Ochman H, Lawrence JG, Groisman EA. 2000. Lateral gene transfer and Appl Environ Microbiol. 71(12):8606–8610. the nature of bacterial innovation. Nature 405(6784):299–304. Swingley WD, et al. 2008. Niche adaptation and genome expansion in the Pinto FL, Thapper A, Sontheim W, Lindblad P. 2009. Analysis of current chlorophyll d-producing cyanobacterium Acaryochloris marina.Proc and alternative phenol based RNA extraction methodologies for cya- Natl Acad Sci U S A. 105:2005–2010. nobacteria. BMC Mol Biol. 10:79. Triglia T, Foote SJ, Kemp DJ, Cowman AF. 1991. Amplification of the Puigbo  P, Wolf YI, Koonin EV. 2010. The tree and net components of multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen prokaryote evolution. Genome Biol Evol. 2:745–756. as multiple independent events. Mol Cell Biol. 11:5244. Qian W, Liao B-Y, Chang AY-F, Zhang J. 2010. Maintenance of duplicate von Rozycki T, Nies DH. 2009. Cupriavidus metallidurans: evolution of a genes and their functional redundancy by reduced expression. Trends metal-resistant bacterium. Antonie Van Leeuwenhoek 96(2): Genet. 26(10):425–430. 115–139. Ratledge C, Dover LG. 2000. Iron metabolism in pathogenic bacteria. Weber M, et al. 2007. Distribution, silencing potential and evolutionary Annu Rev Microbiol. 54:881–941. impact of promoter DNA methylation in the human genome. Nat Reams AB, Neidle EL. 2003. Genome plasticity in Acinetobacter: Genet. 39:457–466. new degradative capabilities acquired by the spontaneous ampli- Yang F, et al. 2010. Biosequestration via cooperative binding of copper by fication of large chromosomal segments. Mol Microbiol. 47(5): Ralstonia pickettii. Environ Technol. 31:1045–1060. 1291–1304. Associate editor: Takashi Gojobori 1492 Genome Biol. Evol. 10(6):1484–1492 doi:10.1093/gbe/evy099 Advance Access publication May 29, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/6/1484/5020734 by Ed 'DeepDyve' Gillespie user on 16 June 2018

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