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Quantitative Transcriptomics Reveals the Growth- and Nutrient-Dependent Response of a Streamlined Marine Methylotroph to Methanol and Naturally Occurring Dissolved Organic Matter

View Article: PubMed Central - PubMed

ABSTRACT

The members of the OM43 clade of Betaproteobacteria are abundant coastal methylotrophs with a range of carbon-utilizing capabilities. However, their underlying transcriptional and metabolic responses to shifting conditions or different carbon substrates remain poorly understood. We examined the transcriptional dynamics of OM43 isolate NB0046 subjected to various inorganic nutrient, vitamin, and carbon substrate regimes over different growth phases to (i) develop a quantitative model of its mRNA content; (ii) identify transcriptional markers of physiological activity, nutritional state, and carbon and energy utilization; and (iii) identify pathways involved in methanol or naturally occurring dissolved organic matter (DOM) metabolism. Quantitative transcriptomics, achieved through addition of internal RNA standards, allowed for analyses on a transcripts-per-cell scale. This streamlined bacterium exhibited substantial shifts in total mRNA content (ranging from 1,800 to 17 transcripts cell−1 in the exponential and deep stationary phases, respectively) and gene-specific transcript abundances (>1,000-fold increases in some cases), depending on the growth phase and nutrient conditions. Carbon metabolism genes exhibited substantial dynamics, including those for ribulose monophosphate, tricarboxylic acid (TCA), and proteorhodopsin, as well as methanol dehydrogenase (xoxF), which, while always the most abundant transcript, increased from 5 to 120 transcripts cell−1 when cultures were nutrient and vitamin amended. In the DOM treatment, upregulation of TCA cycle, methylcitrate cycle, vitamin, and organic phosphorus genes suggested a metabolic route for this complex mixture of carbon substrates. The genome-wide inventory of transcript abundances produced here provides insight into a streamlined marine bacterium’s regulation of carbon metabolism and energy flow, providing benchmarks for evaluating the activity of OM43 populations in situ.

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OM43 strain NB0046 transcription patterns and cell growth assays reveal similar responses to the vitamin (+MeOH +N +V) and HMW DOM (+DOM +N −V) regimes. (A) Hierarchical clustering (with Pearson correlation coefficients and complete linkage) of genome-wide transcript abundances. (B) Maximum NB0046 cell densities in UV-oxidized seawater medium with or without AMS1 vitamin mixture amendment after 72 h of incubation. Error bars are the SDs of biological triplicates. No C Amd., non-carbon-amended control.
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fig6: OM43 strain NB0046 transcription patterns and cell growth assays reveal similar responses to the vitamin (+MeOH +N +V) and HMW DOM (+DOM +N −V) regimes. (A) Hierarchical clustering (with Pearson correlation coefficients and complete linkage) of genome-wide transcript abundances. (B) Maximum NB0046 cell densities in UV-oxidized seawater medium with or without AMS1 vitamin mixture amendment after 72 h of incubation. Error bars are the SDs of biological triplicates. No C Amd., non-carbon-amended control.

Mentions: To quantify the extent of the transcriptional similarity between the vitamin-plus-methanol and HMW DOM regimes further, we used hierarchal clustering to examine how the late-exponential-phase time points from each nutrient regime were related on a global transcriptome scale (Fig. 6A). While the nutrient-deplete (+MeOH −N −V) and nutrient-amended (+MeOH +N −V) regimes formed separate, distinct clusters, the vitamin-amended (+MeOH +N +V) and HMW DOM (+DOM +N −V) regimes clustered together, with the three HMW DOM replicates nested within the vitamin replicates (Fig. 6A). This strong similarity in the global transcriptome indicates that the HMW DOM additions provide vitamins or substituting nutrients that (at least in part) stimulate growth in UV-oxidized seawater. To test this, we conducted an experiment in which vitamins were added to UV-oxidized seawater medium amended with nutrients and looked for recovered growth in the methanol- and non-carbon-amended treatments. In comparison to non-vitamin-amended UV-oxidized seawater, the addition of vitamins stimulated NB0046’s growth in UV-oxidized seawater for both the methanol- and non-carbon-amended treatments (Fig. 6B). This was true for cells grown either in cultivation plates at 1 ml well−1 (Fig. 6B) or in 8-ml cultures in test tubes (data not shown). However, in the test tubes, we continued to see that methanol addition led to a substantial increase in cell concentrations over the HMW DOM addition when vitamins were added, in contrast to the cultivation plates in which the addition of vitamins to the UV-oxidized seawater led to maximum cell density for the HMW DOM amendments.


Quantitative Transcriptomics Reveals the Growth- and Nutrient-Dependent Response of a Streamlined Marine Methylotroph to Methanol and Naturally Occurring Dissolved Organic Matter
OM43 strain NB0046 transcription patterns and cell growth assays reveal similar responses to the vitamin (+MeOH +N +V) and HMW DOM (+DOM +N −V) regimes. (A) Hierarchical clustering (with Pearson correlation coefficients and complete linkage) of genome-wide transcript abundances. (B) Maximum NB0046 cell densities in UV-oxidized seawater medium with or without AMS1 vitamin mixture amendment after 72 h of incubation. Error bars are the SDs of biological triplicates. No C Amd., non-carbon-amended control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120137&req=5

fig6: OM43 strain NB0046 transcription patterns and cell growth assays reveal similar responses to the vitamin (+MeOH +N +V) and HMW DOM (+DOM +N −V) regimes. (A) Hierarchical clustering (with Pearson correlation coefficients and complete linkage) of genome-wide transcript abundances. (B) Maximum NB0046 cell densities in UV-oxidized seawater medium with or without AMS1 vitamin mixture amendment after 72 h of incubation. Error bars are the SDs of biological triplicates. No C Amd., non-carbon-amended control.
Mentions: To quantify the extent of the transcriptional similarity between the vitamin-plus-methanol and HMW DOM regimes further, we used hierarchal clustering to examine how the late-exponential-phase time points from each nutrient regime were related on a global transcriptome scale (Fig. 6A). While the nutrient-deplete (+MeOH −N −V) and nutrient-amended (+MeOH +N −V) regimes formed separate, distinct clusters, the vitamin-amended (+MeOH +N +V) and HMW DOM (+DOM +N −V) regimes clustered together, with the three HMW DOM replicates nested within the vitamin replicates (Fig. 6A). This strong similarity in the global transcriptome indicates that the HMW DOM additions provide vitamins or substituting nutrients that (at least in part) stimulate growth in UV-oxidized seawater. To test this, we conducted an experiment in which vitamins were added to UV-oxidized seawater medium amended with nutrients and looked for recovered growth in the methanol- and non-carbon-amended treatments. In comparison to non-vitamin-amended UV-oxidized seawater, the addition of vitamins stimulated NB0046’s growth in UV-oxidized seawater for both the methanol- and non-carbon-amended treatments (Fig. 6B). This was true for cells grown either in cultivation plates at 1 ml well−1 (Fig. 6B) or in 8-ml cultures in test tubes (data not shown). However, in the test tubes, we continued to see that methanol addition led to a substantial increase in cell concentrations over the HMW DOM addition when vitamins were added, in contrast to the cultivation plates in which the addition of vitamins to the UV-oxidized seawater led to maximum cell density for the HMW DOM amendments.

View Article: PubMed Central - PubMed

ABSTRACT

The members of the OM43 clade of Betaproteobacteria are abundant coastal methylotrophs with a range of carbon-utilizing capabilities. However, their underlying transcriptional and metabolic responses to shifting conditions or different carbon substrates remain poorly understood. We examined the transcriptional dynamics of OM43 isolate NB0046 subjected to various inorganic nutrient, vitamin, and carbon substrate regimes over different growth phases to (i) develop a quantitative model of its mRNA content; (ii) identify transcriptional markers of physiological activity, nutritional state, and carbon and energy utilization; and (iii) identify pathways involved in methanol or naturally occurring dissolved organic matter (DOM) metabolism. Quantitative transcriptomics, achieved through addition of internal RNA standards, allowed for analyses on a transcripts-per-cell scale. This streamlined bacterium exhibited substantial shifts in total mRNA content (ranging from 1,800 to 17 transcripts cell−1 in the exponential and deep stationary phases, respectively) and gene-specific transcript abundances (>1,000-fold increases in some cases), depending on the growth phase and nutrient conditions. Carbon metabolism genes exhibited substantial dynamics, including those for ribulose monophosphate, tricarboxylic acid (TCA), and proteorhodopsin, as well as methanol dehydrogenase (xoxF), which, while always the most abundant transcript, increased from 5 to 120 transcripts cell−1 when cultures were nutrient and vitamin amended. In the DOM treatment, upregulation of TCA cycle, methylcitrate cycle, vitamin, and organic phosphorus genes suggested a metabolic route for this complex mixture of carbon substrates. The genome-wide inventory of transcript abundances produced here provides insight into a streamlined marine bacterium’s regulation of carbon metabolism and energy flow, providing benchmarks for evaluating the activity of OM43 populations in situ.

No MeSH data available.


Related in: MedlinePlus