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Growth Rate-Dependent Coordination of Catabolism and Anabolism in the Archaeon Methanococcus maripaludis under Phosphate Limitation

ABSTRACT: Catabolic and anabolic processes are finely coordinated in microbes to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits identifies fundamental microbial strategies of acclimation. Here, we characterized the systems-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different growth rates under phosphate (i.e., anabolic) limitation. We observed a decoupling of catabolism and anabolism resulting in lower biomass yield relative to catabolically limited cells. In addition, the mass abundance of several coarse-grained proteome sectors exhibited a linear relationship with growth rate, most notably ribosomes and their biogenesis. Accordingly, cellular RNA content also correlated with growth rate. Although the methanogenesis proteome sector was invariant, the metabolic capacity for methanogenesis correlated with growth rate suggesting translationally independent regulation. These observations are in stark contrast to the physiology of M. maripaludis under formate (i.e., catabolic) limitation, where cells keep an invariant proteome including ribosomal content and a high methanogenesis capacity across a wide range of growth rates. Our findings reveal that although highly niche-specialized, M. maripaludis employs fundamentally different strategies to coordinate global physiology during anabolic phosphate and catabolic formate limitation.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Methanococcus Maripaludis S2 (ncbitaxon:267377)

SUBMITTER: Alfred M. Spormann James R. Williamson

PROVIDER: MSV000087621 | MassIVE | Mon Jun 14 20:32:00 BST 2021

SECONDARY ACCESSION(S): PXD026700

REPOSITORIES: MassIVE

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Project description:Hydrogenotrophic methanogenic Archaea are defined by a H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. Hydrogenotrophs respond to H2 starvation both phenotypically and at the level of gene expression; however, the responses during growth on formate have not been characterized. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess, suggesting that energy spilling occurs. Using microarray analysis, we show that the expression of genes encoding F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation, but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess. The strain was grown by continuous culture in a one-liter fermenter (New Brunswick Scientific, Edison, NJ) at 37M-BM-0C (FEMS Microbiol Lett 238: 85-91, 2004). Medium and gas compositions were modified from those for non-limiting conditions (BMC Microbiol 9: 149, 2009). The medium contained 380 mM sodium formate in place of NaCl. The agitation rate was 250 revolutions per minute. The gas mixture initially supplied to the chemostat was H2/Ar/CO2/1% H2S (21/125/40/14 mLs min-1) (Proc Natl Acad Sci USA 104: 8930-8934, 2007). After the OD increased above 0.6 (24 h), the medium flow was turned on at 0.083 L/h. Medium was either non-limiting (described above), formate limiting (modified to contain 200 mM sodium formate and 180 mM NaCl), nitrogen limiting (2 mM NH4Cl), or phosphate limiting (0.08 mM K2HPO4). Also at this point, gas flow through the vessel was shut off, a 1 psi check valve was installed on the exhaust line to maintain positive pressure within the vessel while also allowing for exhaust of gaseous metabolic byproducts, and a 1% Na2S9H2O feed was started into the vessel at 0.83 mL/h. 48 hours later, samples were taken for physiological measurements or microarray analysis. Samples were collected for microarray analysis as follows: 1.5 mLs of chemostat culture (OD660 ~0.65) was centrifuged at 13,000 x g for 30 seconds and supernatant was discarded. The pellet was immediately placed in a dry ice/ethanol bath and stored at -80C until RNA extraction and microarrays could be performed. Total RNA from each sample was compared against a reference RNA pool that was generated in bulk from a mid-log phase culture of MM901. Total RNA from samples and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. After hybridization and washing according to array manufacturer's instructions, the arrays were scanned by Microarray Scanner (Agilent Technologies, Santa Clara, CA). Dye-flip experiments were done for each sample.

Project description:Purpose: Compare the transcriptomes of T. paralvinellae using RNA-Seq gene expression analyses to understand the physiology of this organism when it is grown on different carbon sources, under optimal and hydrogen stress conditions. Methods: T. paralvinellae was grown in triplicate at with either 0.5% maltose or 0.5% tryptone to investigate its catabolic pathways when grown on a sugar and peptides, respectively. It was also grown on 1% formate to determine the effect of formate on cell growth, metabolite production, and gene expression. It was also grown separately on maltose or tryptone media as before, except with hydrogen in the media to examine the effects of hydrogen inhibition. The cells remaining in the 2-liter bioreactor were concentrated by centrifugation. The resulting cell pellets were resuspended in TRIzol (Invitrogen) and total RNA was extracted using a Direct-zol RNA extraction kit (Zymo). RNA quantity was determined with Qubit fluorometry. RNA integrity was checked by a bioanalyzer and a Nanodrop spectrometer. Genewiz sequencing facilities performed rRNA removal, library construction, multiplexing and sequence the RNA with HiSeq2500, 2×100 Paired-End. We aligned the RNAseq reads to T. paralvinellae genome (STAR) and assigned aligned sequence reads to genomic features (featureCounts). We then quantified transcriptome and reported pairwise significant genes that are differentially expressed by DESeq available under Bioconductor (www.bioconductor.org) on Galaxy platform and in R. Results: Sequencing depths ranged from 29,974,378 to 45,240,342 sequences, with a mean of 38,990,002 and a median of 38,951,352 reads per sample. Of 2,138 genes annotated in the T. paralvinellae genome, 2084 transcripts were detected by RNA-Seq.Of the 2084 genes detected by transcriptomics, hydrogen stress caused only 48 genes to be differentially regulated (log2FC > 1, P < 0.05) with maltose and tryptone carbon sources. These included mostly genes associated with formate-dependent metabolism and transporters. The upregulated genes upon growth under H2 stress conditions indicate intrinsic formate production via formate hydrogen lyase and format secretion.

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Growth Rate-Dependent Coordination of Catabolism and Anabolism in the Archaeon Methanococcus maripaludis under Phosphate Limitation

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