Transcriptomic and Physiological Insights into the Robustness of LongFilamentous Cells of Methanosaeta harundinacea, Prevalent in UpflowAnaerobic Sludge Blanket Granules

Liguang Zhou,a Haiying Yu,b Guomin Ai,a Bo Zhang,a Songnian Hu,b Xiuzhu Donga

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of Chinaa; CAS Key Laboratory of GenomeScience and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, People’s Republic of Chinab

Methanosaeta spp. are widely distributed in natural environments, and their filamentous cells contribute significantly to sludgegranulation and the good performance of anaerobic reactors. A previous study indicated that Methanosaeta harundinacea 6Acdisplays a quorum sensing-regulated morphological transition from short to long filaments, and more acetate is channeled intomethane production in long filaments, whereas more is channeled into biomass synthesis in short filaments. Here, we performedtranscriptomic and physiological analysis to gain insights into active methanogenesis in long filaments of M. harundinacea 6Ac.Both RNA sequencing (RNA-seq) and quantitative reverse transcription-PCR indicated that transcription of the genes involvedin aceticlastic methanogenesis and energy metabolism was upregulated 1.2- to 10.3-fold in long filaments, while transcription ofthe genes for the methyl oxidative shunt was upregulated in short filaments. [2-13C]acetate trace experiments demonstrated thata relatively higher portion of the acetate methyl group was oxidized to CO2 in short filaments than in long filaments. The longfilaments exhibited higher catalase activity and oxygen tolerance than the short ones, which is consistent with increased tran-scription of the oxidant-scavenging genes. Moreover, transcription of genes for cell surface structures was upregulated in thelong filaments, and transmission electron microscopy revealed a thicker cell envelope in the filaments. RNA-seq determined a>2-fold upregulation of a variety of antistress genes in short filaments, like those encoding chaperones and DNA repair systems,which implies that the short filaments can be stressed. This study reveals the genetic basis for the prevalence of the long filamen-tous morphology of M. harundinacea cells in upflow anaerobic sludge blanket granules.

Methanogenic degradation of organic complexes is a widelyused approach in wastewater treatment; it promotes the de-

velopment of upflow anaerobic sludge blanket (UASB) reactors(1). Efficient mineralization of organic complexes to CH4 andCO2 in the UASB reactor is implemented by diverse microbesthrough a food chain mode, where methanogens implement thefinal chemical reaction producing CH4 (2, 3). In UASB reactorsand other ecosystems, acetate-derived methane (aceticlasticmethanogenesis) contributes about 70% of the methane pro-duced by either the generalist aceticlastic methanogens of the spe-cies Methanosarcina or the obligate aceticlastic methanogens ofthe species Methanosaeta (4, 5). Methanosaeta species are believedto be the key components in anaerobic digesters not only becauseof their ability to use very low concentrations of acetate (threshold, 5to 20 �M) (6) but also because of their fiber-like cells that serve as ascaffold for the attachment of other organisms to promote the forma-tion of UASB granules (7), an essential self-immobilized organizationof the functional microbes. It has been reported that Methanosaetacells comprise one-third of the anaerobic migrating blanket reac-tor (AMBR) granule biomass (7).

Methanosaeta harundinacea 6Ac was isolated from a UASB re-actor treating beer manufacturing wastewater in Beijing, China(8). When isolated, strain 6Ac cells were long and filamentous, butshort filaments grew upon subculturing in the laboratory. Later,we demonstrated that this cell morphology transition is regulatedby quorum sensing (9), probably through the FilI/FilR two-com-ponent signal transduction system (10). We found that more sub-strate acetate is channeled into cell biomass synthesis in the shortfilaments, while more is channeled into methane production inthe long filaments; the latter has a lower Ks value and a higher Vmax

value in acetate conversion than the short filaments (9). Theseobservations indicate that the long filamentous morphology ofstrain 6Ac cells is the more active cell form in methanogenesis, yetthe genetic basis that enables the robustness of the long filamentsremains unclear.

In this study, through RNA sequencing (RNA-seq)-basedcomparative transcriptomic analysis and assays of relevant physi-ological characteristics, we attempted to identify the gene profilesrelated to robust methanogenesis by long filamentous cells, thepredominant UASB morphology of Methanosaeta strain 6Ac. Ourresults indicated that transcription of the genes responsible foraceticlastic methanogenesis, energy metabolism, and ribosomeproteins was upregulated in the long filaments, which also exhib-ited a higher tolerance for oxidative stress and an enhanced reac-

Received 20 September 2014 Accepted 11 November 2014

Accepted manuscript posted online 14 November 2014

Citation Zhou L, Yu H, Ai G, Zhang B, Hu S, Dong X. 2015. Transcriptomic andphysiological insights into the robustness of long filamentous cells ofMethanosaeta harundinacea, prevalent in upflow anaerobic sludge blanketgranules. Appl Environ Microbiol 81:831–839. doi:10.1128/AEM.03092-14.

Editor: M. A. Elliot

Address correspondence to Xiuzhu Dong, dongxz@im.ac.cn.

L.Z. and H.Y. contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03092-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03092-14

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tive oxygen species (ROS)-scavenging ability, while in the shortfilaments, which appeared to be stressed, both transcriptomic andisotope tracing analysis determined that the methyl oxidativeshunt was upregulated.

MATERIALS AND METHODSMethanogen strain and culture conditions. Methanosaeta harundinacea6AcT (JCM 13211, CGMCC 1.5026, and DSM 17206) was preserved inour laboratory. A previously described prereduced mineral medium con-taining 100 mM sodium acetate was used for routine cultures, and 150 mlculture was dispensed into a 250-ml anaerobic bottle under an atmo-sphere of N2-CO2 (4:1) (8, 9, 11). To culture the long filaments with �10cells inside one sheath, 10% of the late-logarithmic-phase short-filamentculture grown in 50 mM acetate was used as the inoculum, because of thehigh levels of acyl homoserine lactones (AHLs) in this phase. The shortfilaments (�10 cells inside one shell) were cultured by inoculating 10% ofthe same short-filament culture in the stationary phase, when AHL levelsare low. All cultures were incubated at 37°C, unless indicated otherwise.

Total RNA extraction for RNA-seq. Long and short strain 6Ac fila-ments were harvested in the early-mid-log-phase from a 165-ml culture(Fig. 1C) by centrifugation at 13,400 � g for 15 min at 4°C. The cell pelletswere quickly frozen in liquid N2 and then stored at �80°C. Total RNA wasextracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) ac-cording to the manufacturer’s instructions with minor modifications, asfollows. Cell breakage was carried out with 5 cycles of 1 min each in amini-bead beater (Biospec Products, Bartlesville, OK, USA) at 4.2 kHzand quickly frozen in liquid nitrogen. Next, the mixture was centrifuged at13,400 � g at 4°C for 10 min, and the supernatant was moved into RNase-free tubes. Total RNA was then extracted with phenol-chloroform andprecipitated with isopropanol as previously described (12). RNA qualityand quantity were determined on a 1% agarose gel, and 1 unit of RNase-free DNase I and 50 units of RNase inhibitor (Invitrogen) were then addedto 2 �g RNA to digest the remaining chromosomal DNA. This was per-

formed at 37°C for 8 h, and removal of contaminated DNA was thenverified by PCR.

Ion Torrent sequencing and mapping. Five micrograms of total RNAwas again treated with DNase I to remove DNA in either RNase-free wateror Tris-EDTA buffer. rRNAs were depleted with Ribo-Zero RNAs (Epi-centre, Illumina Company, San Diego, CA, USA). A sequencing librarywas constructed following the standard protocols of the Ion Torrent se-quencing technology (Life Technologies).

Two single-end strand-specific libraries were generated for the RNAsextracted from the long and short filaments, which contained 3,320,287and 2,686,614 high-quality reads, respectively. To test their reproducibil-ity, two additional libraries, L (long filaments) and S (short filaments),were prepared from biological replicates in the early mid-log phase. Thebiological replicate libraries generated 2,635,191 (L library) and 2,224,565(S library) high-quality reads. The RNA-seq reads were aligned to the M.harundinacea 6Ac genome using BWA software (13) with four mis-matches and the flag is “-l 8 –O 0 -E 0 -o 3.” Reads mapped to rRNA as wellas those not mapped or mapped to multiple positions by use of theseparameters were discarded. This yielded 2,166,126 and 2,187,758 uniquemapped reads for the long- and short-filament RNAs, respectively, and1,922,658 and 1,488,560 reads from the two biological replicates (L and Slibraries, respectively). These reads accounted for over 70� sequence cov-erage of the entire genome. On the basis of the alignment information,custom Perl scripts were used to calculate transcript coverage depth in-formation.

Analysis of differential transcript abundance. Unique mapped readswere used for transcript abundance analysis in both samples and the bio-logical replicates. Transcript abundance for a gene was evaluated by nor-malization of the read counts to the total mapped unique reads and genelength with the reads per kilobase per million mapped reads (RPKM)function (14). Differences in transcription were identified using theDEGseq package (15) according to the mapped reads count. Those withsignatures considered true (P � 0.001) were noted to be differentially

FIG 1 M. harundinacea 6Ac cell morphology and methane production. (A and B) Epifluorescence microscopic images of the long (A) and short (B) filamentswere taken at 420 nm. Bars � 10 �m. (C and D) Rates of methane production from acetate were determined for growing batch cultures (C) and resting cells (D)of the short (�) and long (�) filaments. Data are the means and standard deviations from three biological replicates of each culture. Arrow, the sampling pointfor RNA-seq.

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transcribed genes, and those with fold changes of �2 or �0.5 were definedto be either up- or downregulated genes, respectively.

Operon definition and gene reannotation. Operons were defined onthe basis of the continuous transcript sequence coverage that extendedinto a codirectional upstream gene (16) and were predicted with ourscript. On the basis of the transcript coverage data, 12 misannotated genesof the automatic annotation were corrected (see Table S1 in the supple-mental material); in these cases, the predicted translation start site oc-curred upstream of the actual transcription start site.

Quantitative reverse transcription-PCR (qRT-PCR). RNA wasquantified by a NanoDrop ND-1000 spectrophotometer (Thermo FisherScientific, Bremen, Germany). Using random primers, the cDNAs wereprepared with a GoldScript cDNA synthesis kit (Invitrogen) according tothe manufacturer’s instructions. Quantitative PCR (qPCR) experimentswere carried out in a Mastercycler ep realplex2 S apparatus (Eppendorf,Hamburg, Germany) using Thunderbird SYBR qPCR mix (Toyobo,Osaka, Japan) as a reporter dye. The real-time PCR oligonucleotide prim-ers (Table 1) were designed using Beacon Designer (version 7.0) software(Premier Biosoft, Palo Alto, CA, USA) to gain maximum amplificationefficiency and sensitivity. For quantification of the transcript abundancefor a gene, a DNA fragment containing the target gene was amplified by

PCR and quantified on the NanoDrop ND-1000 spectrophotometer.These fragments were then serially 10-fold diluted (10�2 to 10�9) in trip-licate and used to generate a standard curve for quantification. The 16SrRNA gene copy number in the cell was used as a reference. Quantificationof a gene transcript was normalized against the 16S rRNA gene copynumbers. The qPCR program was as follows: 1 cycle of 95°C for 30 s andthen 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s (17).Eighteen differentially transcribed genes (see Table S2 in the supplementalmaterial) were tested to verify the RNA-seq data.

Determination of resting-cell methane production rates. Twobatches each of long and short filaments were harvested during their mid-log growth phases by centrifugation at 5,000 � g for 10 min under anaer-obic conditions. Prior to methane production rate determination, thetotal cell proteins of both types of cells were measured. Cell pellets werewashed twice with 0.1 M potassium phosphate buffer (phosphate-buff-ered saline [PBS]; pH 7.2) and resuspended in 0.5 ml PBS. Cells were lysedby ultrasonication at 260 W in a cycle of 1 s sonication and 2 s pause for 20min. Total protein was quantified with the Coomassie protein assay re-agent (Thermo Fisher Scientific). A standard curve for protein concentra-tion was generated with lysozyme.

Cells at an equivalent of 10.8 mg protein were collected and washedthree times with anaerobic washing buffer (pH 7.2, containing KH2PO4,0.545 g; Na2HPO4, 1.336 g; NaCl, 0.234 g; cysteine-HCl, 0.250 g; andNaHCO3, 3.20 g, per liter deionized water under a gas phase of N2-CO2

[80:20]). Cell pellets were suspended in 2 ml anaerobic washing buffercontaining 200 mM sodium acetate in anaerobic tubes and were then usedto measure methane production. Methane production from acetate wasdetermined for each of the three long and short-filament biological repli-cates at 37°C. Methane was determined on a GC-14B gas chromatograph(Shimadzu, Kyoto, Japan) as described by Ma et al. (8). All procedureswere performed anaerobically unless indicated otherwise.

CE preparation. Long- and short-filament cell extracts (CEs) wereprepared as described previously, with modifications (18, 19). Briefly,cultures were harvested from mid-log-phase cells by centrifugation at5,000 � g at 4°C for 15 min, and the cell pellets were washed twice withchilled 0.1 M PBS (pH 7.2). Cell pellets were moved to sterile, precooled1.5-ml Eppendorf tubes, and 0.5 ml PBS containing 0.2 mM phenylmeth-ylsulfonyl fluoride was then added. Cell pellets were lysed by ultrasonica-tion at 240 W in a cycle of 1 s sonication and 2 s pause for 15 min. The celllysate was centrifuged at 13,400 � g at 4°C for 15 min to precipitate the celldebris, and the supernatant (CE) was collected in a precooled Eppendorftube and kept on ice. Total protein was quantified as described above.

Incorporation of [2-13C]acetate. [2-13C]acetate was added to the rou-tinely cultured long and short filaments of M. harundinacea 6Ac at a finalconcentration of 2% (wt/wt) total acetate. Cultures without isotope-la-beled acetate were included as controls. Both cultures were prepared intriplicate. Acetate was determined with a GC-14B gas chromatograph(Shimadzu) with previously reported parameters (11). The stable isotopecomposition was determined with a Trace GC Ultra gas chromatograph(Thermo Fisher Scientific, Milan, Italy) which was connected to a Delta VAdvantage isotope ratio mass spectrometer with a GC Isolink combustionreactor interface (Thermo Fisher Scientific, Bremen, Germany) (20).

Fluorescence microscopy method. Fifty microliters of the strain 6Acculture was spread on a glass slide and dried at room temperature. Thefluorescence at a 420-nm wavelength was examined under a Leica DMI3000 B microscope (Leica Microsystems GmbH, Wetzlar, Germany).

TEM. Long and short filaments were harvested in the late-log growthphase, and samples for transmission electron microscopy (TEM) wereprepared as described previously, with slight modifications (21). Cell pel-lets were fixed with 2.5% glutaraldehyde overnight, followed by postfix-ation in 1% osmium tetroxide for 2 h; after dehydration and infiltration,samples were embedded in Spi-pon 812 resin (Spi Supplies, West Chester,PA, USA), polymerized, and then sectioned with a Leica EM UC6 ultra-microtome (Leica Microsystems GmbH) (22). The ultrathin sections wereapproximately 70 nm thick. They were stained with uranyl acetate and

TABLE 1 Oligonucleotide primers used for qRT-PCR in this study

Primera Sequence (5=¡ 3=)16S rDNAF CCGTGATTGGTGCCGTAGG16S rDNAR CTTCAGCCTGACCTTCATATTGCMhar_0135F ATTCTCCACCAGCAATCAMhar_0135R AGGTCGAGCTTCTTGAACMhar_0376F CTACGACGCCCTGGGAATACMhar_0376R CTCTTAGCCGAGACGACGACMhar_0495F CTCCTTCTTCAGCCACTCCATCMhar_0495R GAACTCCTTGATCTCTCCGTAGACMhar_0689F GATGGACGGAAGGTCAGGAMhar_0689R CGGATGGTCGCATAAGGCMhar_0751F CTTCACCTCCGATGGCTTCTACMhar_0751R AGACATCTTTGGCGTCGTTCCMhar_0793F GAAGTGGTGTCCGACCTTMhar_0793R TCTTCCGTGAACCTGTTGATMhar_0856F AAATGAGCCCGCCCTGAAGMhar_0856R CGTCTCGGTTGTTGTAGTGATCMhar_1288F CCCTTTCACGGCTACCTCAGMhar_1288R GGCAGGCAGCTCGAAGTCMhar_1470F TCCGTCAGGCTCGTTCAGGMhar_1470R CTTGGCTCGGGCGTAGGGMhar_2091F ATTCTCCTGGTGGTTATCMhar_2091R TAGATGTAGACCCAGTTTCMhar_2096F AAGATCGGCAGAGATATAGGMhar_2096R CCGCCAGCAAATAGTATGMhar_2174F CTCCAACGACGCCATCATCTACMhar_2174R CCGCACTCCTTGAAGGTCTTGMhar_2214F ATCTGCAACATGAGCTTCAAGAACMhar_2214R CCCGTATCGAACTGCTTCTCCMhar_2255F CGTGATGATGACCGCCGACTCMhar_2255R TCCTTCCTCACCAGGCATCTCTTCMhar_2322F AGACGAGAAGAACCAGAAGAAGTMhar_2322R AATGGAGGCTTGTTGAAGAAGGMhar_2328F TGGATAAGATGGAAGGCAGTTTTGMhar_2328R TCCTCCACCTCCTTGACTACCMhar_2343F CCGACGCCGCCATAGTATMhar_2343R TTCCGAGACGACGAAGTTCATMhar_2358F AGTATCCCGAGCCCACCTATCTMhar_2358R GGACAGTAGGCGTAGCAGAGGa Each primer is named by the locus to be amplified: F, forward primer; R, reverseprimer.

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lead citrate and observed under an FEI Tecnai Spirit transmission electronmicroscope (FEI, Hillsboro, OR, USA).

Catalase activity assay. Bubble generation was first used to test hydro-gen peroxide scavenging (23) with 25 �l 9.0-mg/ml CEs from both longand short filaments. Catalase activity was determined by the hydrogenperoxide decomposition method (24) with modifications: 65 �l 0.1 mMhydrogen peroxide in PBS and 25 �l 9.0 mg/ml CEs were mixed in anEppendorf tube for 1 min at room temperature. The reaction was stoppedby adding 565 �l stop solution containing 2.5 mM 4-amino-antipyrine(4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one; Sigma, San Fran-cisco, CA, USA) and 0.17 M phenol. After 4 min at room temperature,horseradish peroxidase (Sigma) was added at a final concentration of 50mU/ml. After another 4 min at room temperature, the optical density at510 nm was measured with a Unico 2100 visible-light spectrophotometer(Shanghai, China). A standard curve was generated with chemical H2O2.

Oxygen tolerance assay. The oxygen tolerance of both types of cellswas detected by adding various volumes of air to sterile tubes with 80% N2

and 20% CO2, which generated final oxygen concentrations of 0%, 0.37%,0.74%, and 1.48%; each oxygen concentration was prepared in triplicate.Next, 5-ml cultures in the mid-log phase were added to the air-containingtubes, and methane production was monitored.

RESULTSGenome-wide differentially transcribed genes in long versusshort filaments of M. harundinacea. By inoculating short fila-ments of strain 6Ac in their late log or stationary phase, long fila-ments (Fig. 1A) and short filaments (Fig. 1B) were cultured, re-spectively. When consuming the same quantity of acetate, the longfilaments had a higher methane yield (12.81 � 0.01 mmol) thanthe short filaments (11.86 � 0.01 mmol), while the short filamentsexhibited higher methanogenic rates (0.135 � 0.023 mmol/day)than the long ones (0.105 � 0.047 mmol/day) in the batch culture(Fig. 1C). To differentiate the methane production rates caused bydifferences in cell morphology from those caused by differences inbiomass during growth, the long and short filamentous restingcells (10.8 mg cell protein) were used to determine methane pro-duction from acetate (200 mmol/liter). The results revealed thatthe long filaments exhibited higher methanogenic rates (0.025 �0.000 mmol/day) than the short ones (0.017 � 0.001 mmol/day)(Fig. 1D). This indicates that the higher methanogenic rate ob-served in the short filaments during the growth phase is a result ofa higher biomass.

To examine the gene profiles with differential transcription inlong versus short filaments, high-resolution RNA-seq (Ion Tor-rent) was performed. The comparison indicated that 683 genesshowed transcript abundances that differed by more than 2-foldbetween long and short filaments; of these, 243 and 440 genes wereupregulated in the long and short filaments, respectively (see Ta-ble S3 in the supplemental material).

Furthermore, using qRT-PCR, we quantified the transcriptabundance for 18 genes that differed in their levels of transcriptionin the transcriptomic analysis (see Table S2 in the supplementalmaterial). qRT-PCR and RNA-seq data were well correlated (cor-relation coefficient [r] � 0.810) (see Fig. S1 in the supplementalmaterial), indicating the reliability of the RNA-seq analysis.

Higher levels of transcription of the genes for methanogen-esis and energy metabolism genes in long filaments. Among thegenes with differential transcription, those involved in the aceti-clastic methanogenesis pathway, including acs, cdh, mtr, and mcr,were upregulated 1.5- to 10.3-fold in the long filaments (Fig. 2). Inparticular, two acetyl coenzyme A synthetase genes (Mhar_0749,Mhar_0751), which function in the limiting initial step, increased

their transcript levels 10.1- and 3.8-fold, respectively, and thetranscript abundance of the methyl coenzyme M (methyl-CoM)reductase operon (Mhar_0495 to Mhar_0498) increased 2.2- to10.3-fold in the long filaments. Moreover, the levels of transcrip-tion of the F420 H2 dehydrogenase operon (Mhar_1410 toMhar_1420), which is involved in oxidative phosphorylation, andthe vacuolar type H-ATP synthase operon (Mhar_2253 toMhar_2261) were also increased in the long filaments comparedto their levels of transcription in the short filaments (Table 2). Theenhanced transcription of genes involved in energy metabolism inthe long filaments provides genetic evidence for the robustness oflong filamentous cell morphology.

Coenzyme M-coenzyme B heterodisulfide reductase (Hdr) iskey to the cellular supply of coenzyme M, a compound essentialfor the reduction of methyl to CH4. Two types of hdr operons arepresent in the M. harundinacea 6Ac genome, and hdrED(Mhar_0792, 0793) transcription was upregulated in long fila-ments. The level of expression of the membrane-bound hdrD genewas approximately 9.3-fold higher than that of hdrABC (Fig. 2) atthe transcription level. This suggests that HdrED plays a majorrole in aceticlastic methanogenesis in M. harundinacea 6Ac. Ad-ditionally, most of the ribosome-encoding genes were upregu-lated in long filaments (see Table S4 in the supplemental material).

However, expression of some genes in the methyl oxidationshunt, such as the tungsten formylmethanofuran dehydrogenaseoperon (fwd), was higher in short filaments than in long filamentsat the mRNA level (Fig. 2). qRT-PCR also determined a 2-foldupregulation of a gene for the F420 redox reaction (Mhar_2358)involved in the methyl oxidative shunt in short filaments (seeTable S2 in the supplemental material), implying that more reduc-ing equivalents are gained in short filaments than in long fila-ments. Moreover, genes involved in gluconeogenesis were up-regulated at the mRNA levels in short filaments (see Table S5 inthe supplemental material). This indicates that biomass synthesisis more active in short filaments than in long filaments, which isconsistent with previous findings (9).

The methyl oxidative pathway is active in short filaments. Tocompare methyl oxidative shunt activity in both types of cells,2-13C-labeled acetate was applied to determine 13CO2 productionfrom the methyl group of acetate, which is believed to be reducedto CH4. Upon acetate depletion by incubating strain 6Ac at 37°Cfor 55 days, the 13CO2 yield was higher in short filaments (13CCO2

,�1.87 � 0.05) than in long filaments (13CCO2

, �14.31 � 0.30)(P � 0.01) (Table 3), supporting the idea that the methyl oxidativeshunt is more active in short filaments.

By subtracting the CH4 yield from the consumed acetate, wecalculated that, under the culture conditions used in this study,0.5% and 3.6% of the acetate methyl carbon-formed CO2 wasproduced via the methyl oxidative shunt in the long and shortfilaments, respectively (Table 3). It is believed that the methyloxidative shunt is used to generate reducing equivalents that canbe used for ATP synthesis or cell biomass, because the mch mu-tants of Methanosarcina barkeri and Methanosarcina acetivoranshave lost the ability to grow on acetate (25). Therefore, the isotopetracing experiment supported the different ratios of acetate chan-neled to CH4 production and biomass formation in cells of thetwo morphologies.

Long filaments efficiently scavenge ROS. ROS are lethal toanaerobes because they possess insufficient ROS-scavenging pro-teins; these include superoxide dismutase (SOD) and catalase in

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aerobes and superoxide reductase (SOR) in anaerobes (26, 27). M.harundinacea 6Ac possesses genes encoding catalase, SOD, andother peroxidases but not genes encoding SOR. Some oxidant-scavenging genes were upregulated in long filaments (Table 4) atthe transcription level. This included catalase (Mhar_0135), fla-voprotein (Mhar_0340), thioredoxin (Mhar_1159), and alkylhy-droperoxidase-like protein (Mhar_1400). However, the genes forrubredoxin-type Fe(Cys)4 protein (Mhar_1374) and ferritin(Mhar_1375) were upregulated in short filaments at the transcrip-tion level (Table 4). This implies that cells of the two morphologiesmay exhibit varied potentials in antioxidative stress.

To compare ROS-scavenging activity in both types of cells,catalase activity was detected by adding 0.093 mmol hydrogenperoxide. More bubbles were produced in the long-filament cul-ture than in the short-filament culture. Furthermore, 0.047 �0.002 mmol H2O2 was degraded within 1 min by 0.225 mg CEfrom the long filaments, while only 0.023 � 0.001 mmol H2O2 wasremoved by the same amount of CE from the short filaments. Thisindicates higher oxidant-scavenging activity in the long filaments.

To further determine the oxygen tolerance of both types of M.harundinacea 6Ac cells, various volumes of air were added to thegas phase of the cultures. A growth assay showed that the short

FIG 2 Differential expression of genes involved in aceticlastic methanogenesis and the methyl oxidative shunt in M. harundinacea 6Ac. Bar graphs flanking themethanogenic pathways show the related transcript abundances detected in the transcriptomes of short and long filaments. Red and green, genes upregulated inlong and short filaments, respectively; black, genes for which there was no differential transcription. *, transcription was verified by qRT-PCR. MFR, methane-furan; THMPT, tetrahydromethanopterin; MPT, methanopterin; HS-CoB and CoB, coenzyme B; CoB-S-S-CoM, heterodisulfide of coenzyme M and coenzymeB; HS-CoM, coenzyme M; Acetyl-S-CoA, acetyl-coenzyme A; acs, acetyl coenzyme A synthetase; [CO], CO-carbon monoxide dehydrogenase complex; cdh, COdehydrogenase; Fd, ferredoxin; fmd, molybdenum-formyl-MFR dehydrogenase (Mhar_1283 to Mhar_1288); fwd, tungsten-containing formyl-MFR dehydro-genase (Mhar_0014, Mhar_0373 to Mhar_0376, and Mhar_2308 to Mhar_2310); ftr, formyl-MFR:THMPT formyltransferase (Mhar_2214); mch, N5N10-methenyl-THMPT cyclohydrolase (Mhar_2174); mtd, F420-dependent N5N10-methylene-THMPT dehydrogenase (Mhar_1470); mer, F420-dependent N5N10-methylene-THMPT reductase (Mhar_0856); mtr, N5-methyl-THMPT methyltransferase (Mhar_2090 to Mhar_2097); mcr, methyl-CoM reductase (Mhar_0495to Mhar_0498 and Mhar_0529); hdr, heterodisulfide reductase (Mhar_0604 to Mhar_0606 and Mhar_0792 to Mhar_0793).

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filaments ceased growth when 0.74% oxygen was added to theheadspace, while the long filaments stopped growing with 1.48%oxygen (Fig. 3). Both the enzymatic assay and O2 tolerance furtherconfirmed the higher antioxidative stress capacity of the long fil-aments.

Moreover, we did not observe oxygen-induced expression ofthe genes encoding ROS-scavenging proteins, indicating that theyare not regulated by oxygen, but the quorum sensing-regulatedmorphology alteration is related to oxidant clearance in M. har-undinacea 6Ac.

Short filaments appear to be stressed. As in bacteria and otherarchaea, a gene cluster (Mhar_2125 to Mhar_2127) for chaper-ones (dnaJ, dnaK, and grpE) was present in the M. harundinacea6Ac genome. Transcriptomic analysis indicated that these genes

were upregulated in the short filaments (Table 4). Additionally,two of the three thermosome subunit genes (Mhar_0878 andMhar_2128) that function as protein-folding chaperones and agene encoding the heat shock protein Hsp20 (Mhar_1971) werealso upregulated at the mRNA level in short filaments. Moreover,many genes related to DNA repair were upregulated in short fila-ments at the transcription level (see Table S6 in the supplementalmaterial). These data combined suggest that M. harundinacea 6Acshort filaments, the morphology induced under laboratory con-ditions, were stressed.

Cell envelope synthesis and cell division are enhanced inlong and short filaments, respectively. Epifluorescence micro-scopic images revealed that long filaments of M. harundinacea 6Acconsisted of multiple rod cells wrapped in a single sheath (Fig. 1A).

TABLE 2 Oxidative phosphorylation and biomass synthesis gene transcript abundance in long versus short filaments of M. harundinacea 6Aca

Gene function and locus Annotationc

Fold change inabundance (L/S) Operon

Electron transfers (F420 H2 dehydrogenase)Mhar_0854 FpoO 2.4Mhar_1410 FpoA — Mhar_1410-Mhar_1420Mhar_1411 FpoB —Mhar_1412 FpoD —Mhar_1413 FpoH 1.5Mhar_1414 FpoI —Mhar_1415 FpoJ 2.5Mhar_1416 FpoJ —Mhar_1417 FpoK 1.9Mhar_1418 FpoL 1.3Mhar_1419 FpoM 1.4Mhar_1420 FpoN 1.5

V-type ATP synthase subunitMhar_2253 V-type ATP synthase subunit D 1.8 Mhar_2253-Mhar_2261Mhar_2254 V-type ATP synthase subunit B 2.2Mhar_2255 V-type ATP synthase subunit A 3.5(2.0b)Mhar_2256 V-type ATP synthase subunit F 1.6Mhar_2257 V-type ATP synthase subunit C 2.0Mhar_2258 V-type ATP synthase subunit E 1.2Mhar_2259 H-transporting two-sector ATPase subunit C —Mhar_2260 V-type ATP synthase subunit I 1.9Mhar_2261 V-type ATP synthase subunit H 2.2

a Abbreviations and symbols: L/S, long/short filaments; Fpo, F420 H2 dehydrogenase; —, no difference in transcript abundance.b The fold change in parentheses was that detected by qRT-PCR.c V-type, vacuolar type H-ATP synthase.

TABLE 3 Gas chromatography/isotope ratio mass spectrometry-determined 13CCO2and 13CCH4

in the culture gas phases and total methane andcarbon dioxide yield from the acetate consumed by long and short filaments of M. harundinacea 6Ac

Cell shape and product

13CVPDBa value (‰) of product

Amt of acetateconsumed(mmol)

Amt of CH4 produced(mmol)

Calculated amt ofCO2 produced(mmol)Natural acetateb [2-13C]acetatec

Long filaments 12.28 � 0.15 12.22 � 0.15 0.06 � 0.01CH4 �27.64 � 0.60 1,738.39 � 0.34CO2 �28.42 � 0.09 �14.31 � 0.30

Short filaments 12.30 � 0.13 11.85 � 0.12 0.45 � 0.01CH4 �28.07 � 0.02 1,711.31 � 12.4CO2 �27.54 � 0.07 �1.87 � 0.05

a 13CVPDB, isotope ratios reported as values in per mille deviations from the VPDB scale with a 13C/12C value of 0.0111802.b Substrate acetate is at 100 mmol/liter.c Substrate includes 98 mmol/liter natural acetate and 2 mmol/liter of [2-13C]acetate.

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RNA-seq determined that several genes for cell surface proteins,including an S-layer protein gene (Mhar_0562) and a cell surfaceprotein gene (Mhar_1933), had increased levels of transcription inthe long filaments (see Table S7 in the supplemental material).Additionally, genes encoding ABC transporters for tungstate(Mhar_1363 and Mhar_1364) and molybdate (Mhar_2341 toMhar_2343) also had increased transcript abundance.

Ultrathin sections of long and short filaments were examinedby electronic microscopy to identify the cell structures relevant tothe differential transcription of the cell surface protein genes.

Thicker cell walls and a large intracellular cleat-like structure wereobserved in the long filaments (see Fig. S2 in the supplementalmaterial). The cross-sectional profiles also revealed that 90% oflong filamentous cells were wrapped in an irregular envelope (seeFig. S2B in the supplemental material); this probably resultedfrom the presence of abundant cell surface proteins. Such a struc-ture was infrequently observed in the short filaments (see Fig. S2Din the supplemental material).

RNA-seq revealed that DNA replication and cell division geneswere upregulated in the short filaments of M. harundinacea 6Ac(see Table S8 in the supplemental material). This could be relatedto fast growth in the short filaments.

DISCUSSION

Methanosaeta strains are characterized by their ability to producemethane even from very low concentrations of acetate, which maybe the reason why they are so widely distributed, e.g., in anaerobicwaste digesters, rice paddies, and natural wetlands (28, 29), andare the predominant methane producers in many environments(30). The fiber-like Methanosaeta cells are particularly advanta-geous in the formation of granules, hence their important role inwastewater treatment (31, 32). Previously, we have found that M.harundinacea 6Ac undergoes a quorum sensing-regulated cellmorphology transit from short to long filaments, and the formerchannels more acetate to cell biomass synthesis, while the latterdirects more into methane production (9). This work, throughanalyzing the differentially transcribed gene profiles, isotope trac-ing, and biochemical experiments, reveals the molecular basis ofthe different physiological features between the two types of cells.

CO2 reductive methanogenesis is the only pathway for meth-ane production in the hydrogenotrophic methanogens, while itacts as the methyl oxidative shunt to generate reducing equiva-lents in the methylotrophic methanogens. Smith and Mah re-ported that small quantities of acetate are oxidized to producereductive equivalents for biosynthesis in Methanosarcina sp. strain227 (33). The function of this pathway for generating reductivebiosynthesis has been confirmed in Methanosarcina species grow-ing on acetate through assays of enzymatic activity (5, 34). It hasbeen further demonstrated that Methanosarcina mutants with adeletion of mch, a gene encoding N5N10-methenyl-tetrahydro-methanopterin cyclohydrolase in the methyl oxidative shunt, failto grow in acetate (25). Though implementing obligate aceticlasticmethanogenesis, Methanosaeta spp. carry a suite of genes for theCO2-reductive methanogenesis pathway in their genomes.Through molecular and enzymatic studies, as well as 2-13C-la-beled acetate tracing assays, we have determined that this pathwayalso acts as the methyl oxidative shunt in M. harundinacea 6Ac(11). This work demonstrates that the methyl oxidative shunt isactive only in short filaments, which is consistent with the previ-ous finding that the short filaments produce more biomass but lessmethane. Data from RNA-seq and the physiological experimentshow that short filaments of M. harundinacea 6Ac exhibit a lowermethanogenic rate and lower oxidative tolerance than long fila-ments but higher levels of transcription of the chaperone andDNA repair genes than long filaments. Hence, the laboratory-induced short filaments are under stress, while the long ones arehealthier. In Escherichia coli, the filamentous phenotype can over-come host innate immunity, for instance, during urinary tractinfection, as the phenotype can protect cells from the lethal envi-ronment (35). Similarly, long filaments can benefit Methanosaeta

TABLE 4 Transcript abundance of genes encoding stress-relatedproteins in long versus short filaments of M. harundinacea 6Ac

Locus AnnotationFold changea

(L/S)

Mhar_0135 Catalase/peroxidase HPIc 2.1 (2.1b)Mhar_0208 Superoxide dismutase (Mn/Fe) —Mhar_0206 Peroxiredoxin, putative —Mhar_1795 Peroxiredoxin, putative —Mhar_1087 Peroxiredoxin family protein —Mhar_1374 Rubredoxin-type Fe(Cys)4 protein �2.5Mhar_1594 Putative type A flavoprotein —Mhar_1375 Ferritin, Dps family protein �2.6Mhar_1603 Peptide methionine sulfoxide

reductase MsrA—

Mhar_0076 Flavoprotein —Mhar_0340 Flavoprotein 2.3Mhar_1594 Putative type A flavoprotein —Mhar_0183 Rubrerythrin —Mhar_1343 Rubrerythrin —Mhar_1835 Rubrerythrin —Mhar_0226 Thioredoxin reductase —Mhar_1159 Thioredoxin 2.1Mhar_0911 Thioredoxin —Mhar_2116 Rubredoxin —Mhar_1543 Redox-active disulfide protein 2 —Mhar_1723 Desulfoferrodoxin Dfx domain-

containing protein—

Mhar_1400 Alkylhydroperoxidase-like protein,AhpD family

2.6

Mhar_2125 Chaperone protein DnaJ �2.1Mhar_2126 Chaperone protein DnaK �1.8Mhar_2127 Cochaperone GrpE �5.8Mhar_0878 Thermosome subunit �2.3Mhar_1232 Thermosome subunit delta —Mhar_2128 Thermosome subunit �2.5Mhar_2363 Thermosome subunit �1.9Mhar_1811 Heat shock protein HtpX —Mhar_0035 Small heat shock protein �1.7Mhar_0286 Heat shock protein Hsp20 —Mhar_1971 Heat shock protein Hsp20 �5.1Mhar_1165 Peptidyl-prolyl cis-trans isomerase 5.6Mhar_1166 Peptidyl-prolyl cis-trans isomerase �9.5Mhar_1438 Peptidyl-prolyl cis-trans isomerase —Mhar_1804 Peptidylprolyl isomerase, FKBP-type —Mhar_2165 Proteasome subunit alpha —Mhar_2229 Proteasome subunit beta —Mhar_1739 Prefoldin subunit alpha �1.6Mhar_2156 Prefoldin subunit beta 2.6a Abbreviations and symbols: L/S, long/short filaments; —, no difference in transcriptabundance; negative values, the gene was downregulated in long filaments.b The fold change in parentheses was that detected by qRT-PCR.c HPI, hydroperoxidase I.

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species survival in diverse environments, such as those in the ox-idative stressed state.

To test whether quorum sensing-regulated long filaments ofM. harundinacea 6Ac benefit UASB granulation, we carried outexperiments by dosing either M. harundinacea alone or M. harun-dinacea combined with its spent culture containing the quorum-sensing signal molecules into UASB reactors treating syntheticwastewater. After 3 months, we found that quorum sensing-me-diated long filaments of M. harundinacea 6Ac did promote gran-ulation and constant chemical oxygen demand (COD) removalefficiency. Moreover, 16S rRNA homolog analysis detected thepresence of Methanosaeta spp. in the UASB granules and foundthat species diversity was increased in the long filaments of M.harundinacea 6Ac-dominant UASB granules including not onlymethanogen species but also, presumably, functional bacterialspecies, such as syntrophic bacteria (data not shown).

A recent study found that the obligate aceticlastic methanogenM. harundinacea 6Ac even carries out CO2-reductive methano-genesis when it gains electrons from Geobacter metallireducensthrough nanowires (36). This indicates that dormant pathways inmicrobes can be activated when the right triggers are present, thusmaximizing their metabolic potential.

ACKNOWLEDGMENTS

We thank Tong Sun in the State Key Laboratory of Microbial Resourcesfor valuable suggestions on mass spectrum experiments and Yanxia Jiaand Lei Sun in the Center for Biological Imaging, Institute of Biophysics,Chinese Academy of Sciences, for TEM work.

This work was supported by a grant from the National Natural ScienceFoundation of China (31100035).

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