Project description:The genome of the marine Synechococcus sp. WH8102 displays a minimal regulatory network yet physiological and molecular responses of this organism are tuned to episodic limitation for nitrogen and phosphorus. Microarray analyses have demonstrated a key role for the two-component regulatory system, PhoBR, in the regulation of P transport and metabolism in this strain. However, there is some evidence that another regulator, SYNW1019 (designated as ptrA), probably under the control of PhoB, is involved in the wider response to P-depletion. PtrA is one of only two genome encoded DNA binding proteins of the CRP family in Synechococcus sp. WH8102, and a potential transcriptional regulator with homology to NtcA, the global nitrogen regulator in cyanobacteria. To define the precise role of this regulator we constructed a mutant by insertional inactivation and compared the physiology of wild-type Synechcococcus sp. WH8102 with the ptrA mutant under P-replete and P-deplete growth conditions. During P-depletion the ptrA mutant failed to up-regulate phosphatase activity. Microarrays and quantitative RT-PCR indicate that a subset of the Pho regulon is directly controlled by PtrA, including two phosphatase genes (SYNW0196, SYNW2390), a predicted phytase (SYNW0762) and a gene of unknown function (SYNW0165) all of which are highly up regulated during P-limitation. This result was confirmed by electrophoretic mobility shift assays which demonstrated binding of over expressed PtrA to promoter sequences upstream of the induced phosphatases (SYNW0165, SYNW0196 and SYNW2390). This work suggests a two-tiered response to P-depletion in this strain, the first being PhoB-dependent induction of high affinity PO4 transporters, and the second the PtrA-dependent induction of phosphatases for scavenging organic P. The levels of numerous other transcripts are also directly or indirectly controlled by PtrA, including those involved in cell surface modification, metal uptake, photosynthesis, stress responses and other metabolic process, which may indicate a wider role for PtrA in cellular organisation. In an environmental context ptrA is found in a number of picocyanobateria isolated from a range of oceanic provinces, including strains that lack a functional phoBR..These results give broader insight into the regulation of physiological responses that may dictate niche adaptation in genetically diverse lineages of marine Synechococcus, and suggest that signalling networks and coordinated responses to nutrient availability are important, even in oligotrophic ocean environments.

Project description:Characterizing the cell-level metabolic trade-offs that phytoplankton exhibit in response to changing environmental conditions is important for predicting the impact of these changes on marine food web dynamics and biogeochemical cycling. The time-selective proteome-labeling approach, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to provide insight into differential allocation of resources at the cellular level, especially when coupled with proteomics. However, the application of this technique in marine phytoplankton remains limited. We demonstrate that the marine cyanobacteria Synechococcus sp. and two groups of eukaryotic algae take up the modified amino acid l-homopropargylglycine (HPG), suggesting that BONCAT can be used to detect translationally active phytoplankton. However, the impact of HPG addition on growth dynamics varied between groups of phytoplankton. In addition, proteomic analysis of Synechococcus cells grown with HPG revealed a physiological shift in nitrogen metabolism, general protein stress, and energy production, indicating a potential limitation for the use of BONCAT in understanding the cell-level response of Synechococcus sp. to environmental change. Variability in HPG sensitivity between algal groups and the impact of HPG on Synechococcus physiology indicates that particular considerations should be taken when applying this technique to other marine taxa or mixed marine microbial communities. IMPORTANCE Phytoplankton form the base of the marine food web and substantially impact global energy and nutrient flow. Marine picocyanobacteria of the genus Synechococcus comprise a large portion of phytoplankton biomass in the ocean and therefore are important model organisms. The technical challenges of environmental proteomics in mixed microbial communities have limited our ability to detect the cell-level adaptations of phytoplankton communities to a changing environment. The proteome labeling technique, bioorthogonal noncanonical amino acid tagging (BONCAT), has potential to address some of these challenges by simplifying proteomic analyses. This study explores the ability of marine phytoplankton to take up the modified amino acid, l-homopropargylglycine (HPG), required for BONCAT, and investigates the proteomic response of Synechococcus to HPG. We not only demonstrate that cyanobacteria can take up HPG but also highlight the physiological impact of HPG on Synechococcus, which has implications for future applications of this technique in the marine environment.

Project description:The genome of the marine Synechococcus sp. WH8102 displays a minimal regulatory network yet physiological and molecular responses of this organism are tuned to episodic limitation for nitrogen and phosphorus. Microarray analyses have demonstrated a key role for the two-component regulatory system, PhoBR, in the regulation of P transport and metabolism in this strain. However, there is some evidence that another regulator, SYNW1019 (designated as ptrA), probably under the control of PhoB, is involved in the wider response to P-depletion. PtrA is one of only two genome encoded DNA binding proteins of the CRP family in Synechococcus sp. WH8102, and a potential transcriptional regulator with homology to NtcA, the global nitrogen regulator in cyanobacteria. To define the precise role of this regulator we constructed a mutant by insertional inactivation and compared the physiology of wild-type Synechcococcus sp. WH8102 with the ptrA mutant under P-replete and P-deplete growth conditions. During P-depletion the ptrA mutant failed to up-regulate phosphatase activity. Microarrays and quantitative RT-PCR indicate that a subset of the Pho regulon is directly controlled by PtrA, including two phosphatase genes (SYNW0196, SYNW2390), a predicted phytase (SYNW0762) and a gene of unknown function (SYNW0165) all of which are highly up regulated during P-limitation. This result was confirmed by electrophoretic mobility shift assays which demonstrated binding of over expressed PtrA to promoter sequences upstream of the induced phosphatases (SYNW0165, SYNW0196 and SYNW2390). This work suggests a two-tiered response to P-depletion in this strain, the first being PhoB-dependent induction of high affinity PO4 transporters, and the second the PtrA-dependent induction of phosphatases for scavenging organic P. The levels of numerous other transcripts are also directly or indirectly controlled by PtrA, including those involved in cell surface modification, metal uptake, photosynthesis, stress responses and other metabolic process, which may indicate a wider role for PtrA in cellular organisation. In an environmental context ptrA is found in a number of picocyanobateria isolated from a range of oceanic provinces, including strains that lack a functional phoBR..These results give broader insight into the regulation of physiological responses that may dictate niche adaptation in genetically diverse lineages of marine Synechococcus, and suggest that signalling networks and coordinated responses to nutrient availability are important, even in oligotrophic ocean environments. In this series gene expression of a ptrA mutant has been analyzed under phosphorus deplete conditions just after the onset of induction of phosphatase activity in wild type. There are six duel-channel slides upon which both ptrA mutant and wild type samples were hybridized. There are three technical replicates for each of two biological replicates including one flip-dye comparison. Each slide contains six replicate spots per gene.

Project description:The northern Adriatic is highly productive and shallow area characterized by numerous spatio-temporal gradients (e.g. nutrients, salinity, temperature). It is strongly influenced by numerous freshwater inputs, mainly from Po river. Its current systems as well as Po river, generates gradients of phosphate availability with an expressed N/P imbalance and phosphate limitation. A number of recent studies characterized these gradients as major factors affecting abundance and composition of microphytoplankton communities. Focus of this study is on two Leptocylindrus species, Leptocylindrus aporus (F.W. French & Hargraves) D. Nanjappa & A. Zingone 2013 and Leptocylindrus hargravesii D. Nanjappa & A. Zingone 2013. Species belonging to Leptocylindrus genus are frequently observed and have high abundances and also high contributions to the microphytoplankton community in this area. We focused on their morphological and physiological responses to phosphate limitation in situ and also performed in vitro experiments. In this study we report data on species specific growth rates under phosphorus (P) deplete and P rich conditions, localization and characteristics of alkaline phosphate activity, phosphate uptake rates as well as their morphological differences in P deplete versus P rich conditions. Our in vitro experiments showed that both Leptocylindrus species morphologically reacted similarly to phosphorus depletion and showed significantly elongated pervalvar axis in P depleted conditions if compared to P rich conditions. Also average chain lengths increased when in P depleted conditions. Two previously mentioned adaptations indicate their tendency to increase cellular surface areas available for alkaline phosphatase. Chlorophyll fluorescence of both species significantly decreased in P depleted medium. Although both species morphologically reacted similarly, our experiment demonstrated significant differences in physiological reactions to P depleted conditions.

Dataset Information

Influence of growth rate on the physiological response of marine Synechococcus to phosphate limitation.

Publications

Influence of growth rate on the physiological response of marine Synechococcus to phosphate limitation.

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