Genomic and phenotypic differentiation among <i>Methanosarcina mazei</i> populations from Columbia River sediment

Youngblut, Nicholas D.; Wirth, Joseph S.; Henriksen, James R.; Smith, Maria W.; Simon, Holly M.; Metcalf, William W.; Whitaker, Rachel J.

doi:10.1038/ismej.2015.31

Cited by 36 publications

(35 citation statements)

References 66 publications

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“…(35), as was done in Sulfolobus islandicus (36). First, the CRISPR/Cas subtypes vary significantly across the genus Methanosarcina, even within strains belonging to the same species (35).…”

Section: Discussionmentioning

confidence: 99%

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

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Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeon Methanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJdependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homologydirected repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.he CRISPR (clustered regularly interspaced palindromic repeats) array and associated cas genes are widespread in microbial genomes (1), where they confer acquired immunity to phage and foreign DNA elements (2). The type IIA system from Streptococcus pyogenes is especially well characterized and has been widely applied as a remarkably effective genome-editing tool (3). During genome editing, heterologous expression of the RNA-guided DNA endonuclease Cas9 and a chimeric singleguide (sg) RNA, comprised of a 20-bp spacer that targets the chromosome and an 80-bp scaffold that binds Cas9, leads to a lethal double-strand break (DSB) at all target sites within the genome that are flanked by a 3′ NGG protospacer adjacent motif (PAM) (4) (Fig. S1). In eukaryotes, the nonhomologous endjoining (NHEJ) repair pathway can mend the DSB by generating simple insertions or deletions at the sgRNA target site, thus preventing additional rounds of Cas9-mediated cleavage (3, 5). Alternatively, the native homology-dependent repair (HDR) pathway can repair the fatal DSB, so long as a repair template that modifies or removes the sgRNA target site is provided, again preventing additional rounds of Cas9-mediated cleavage (3, 5) (Fig. S1). Appropriately ...

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“…(35), as was done in Sulfolobus islandicus (36). First, the CRISPR/Cas subtypes vary significantly across the genus Methanosarcina, even within strains belonging to the same species (35).…”

Section: Discussionmentioning

confidence: 99%

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Nayak

Metcalf

2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

123

112

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“…Genes that were not identified as differentially expressed could be optimized for the different environments in which M. mazei and M. acetivorans grow; perhaps due to the adaptation to freshwater and saline environments. A recent study of M. mazei strains along the Columbia River show differences in genomic content comparing those in fresh- and salt-water environments that resulted in differences in metabolic efficiency/utilization of TMA [68]. …”

Section: Discussionmentioning

confidence: 99%

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

et al. 2016

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BackgroundWhile a few studies on the variations in mRNA expression and half-lives measured under different growth conditions have been used to predict patterns of regulation in bacterial organisms, the extent to which this information can also play a role in defining metabolic phenotypes has yet to be examined systematically. Here we present the first comprehensive study for a model methanogen.ResultsWe use expression and half-life data for the methanogen Methanosarcina acetivorans growing on fast- and slow-growth substrates to examine the regulation of its genes. Unlike Escherichia coli where only small shifts in half-lives were observed, we found that most mRNA have significantly longer half-lives for slow growth on acetate compared to fast growth on methanol or trimethylamine. Interestingly, half-life shifts are not uniform across functional classes of enzymes, suggesting the existence of a selective stabilization mechanism for mRNAs. Using the transcriptomics data we determined whether transcription or degradation rate controls the change in transcript abundance. Degradation was found to control abundance for about half of the metabolic genes underscoring its role in regulating metabolism. Genes involved in half of the metabolic reactions were found to be differentially expressed among the substrates suggesting the existence of drastically different metabolic phenotypes that extend beyond just the methanogenesis pathways. By integrating expression data with an updated metabolic model of the organism (iST807) significant differences in pathway flux and production of metabolites were predicted for the three growth substrates.ConclusionsThis study provides the first global picture of differential expression and half-lives for a class II methanogen, as well as provides the first evidence in a single organism that drastic genome-wide shifts in RNA half-lives can be modulated by growth substrate. We determined which genes in each metabolic pathway control the flux and classified them as regulated by transcription (e.g. transcription factor) or degradation (e.g. post-transcriptional modification). We found that more than half of genes in metabolism were controlled by degradation. Our results suggest that M. acetivorans employs extensive post-transcriptional regulation to optimize key metabolic steps, and more generally that degradation could play a much greater role in optimizing an organism’s metabolism than previously thought.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3219-8) contains supplementary material, which is available to authorized users.

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“…1B). BLAST searches for homologues in other methanoarchaea detected high sequence conservation, particularly among recently released draft genomes of Methanosarcina species (Youngblut et al, 2015) with 100% identity in the M. mazei strains (Fig. 1C).…”

Section: Srna 41 Characteristicsmentioning

confidence: 97%

sRNA₄₁ affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1

Buddeweg

Sharma

Urlaub

et al. 2018

Molecular Microbiology

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Several noncoding RNAs potentially involved in nitrogen (N)-regulation have been detected in Methanosarcina mazei, however, targets have been identified only for one of them. Here, we report on the function of sRNA , highly expressed under N-sufficiency. Comprising 120 nucleotides, sRNA shows high sequence and structural conservation within draft genomes of numerous Methanosarcina species. In silico target prediction revealed several potential targets, including genes of two homologous operons encoding for acetyl-CoA-decarbonylase/synthase complexes (ACDS) representing highly probable target candidates. A highly conserved single stranded region of sRNA was predicted to mask six independent ribosome binding sites of these two polycistronic mRNAs and was verified in vitro by microscale thermophoresis. Proteome analysis of the respective sRNA -deletion mutant showed increased protein expression of both ACDS complexes in the absence of sRNA , whereas no effect on transcript levels was detected, arguing for sRNA -mediated post-transcriptional fine-tuning of ACDS expression. We hypothesize that the physiological advantage of downregulating sRNA under N-limiting conditions is the resulting increase of ACDS protein levels. This provides sufficient amounts of amino acids for nitrogenase synthesis as well as reducing equivalents and energy for N -fixation, thus linking the carbon and N-metabolism.

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Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment

Cited by 36 publications

References 66 publications

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

sRNA₄₁ affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1

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Genomic and phenotypic differentiation among Methanosarcina mazei populations from Columbia River sediment

Cited by 36 publications

References 66 publications

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

sRNA41 affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1

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sRNA₄₁ affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1