Limits...
Quantitative and functional characterization of the hyper-conserved protein of Prochlorococcus and marine Synechococcus.

Whidden CE, DeZeeuw KG, Zorz JK, Joy AP, Barnett DA, Johnson MS, Zhaxybayeva O, Cockshutt AM - PLoS ONE (2014)

Bottom Line: Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production.The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments.We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Biochemistry, Mount Allison University, Sackville, NB, Canada.

ABSTRACT
A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

Show MeSH

Related in: MedlinePlus

Schematic presentation of the genomic region around the PSHCP gene.Panel A. Mapping of promoters and primers onto the region. In all examined picocyanobacterial genomes the upstream region consists of the ribosomal protein L19 (rpL19) and tryptophanyl-tRNA genes. The locations of predicted promoters are shown by bent arrows, while the locations of the reverse (R1) and forward (F1 to F6) primers used for RT-Q-PCR analysis are indicated by half arrows (see Table S1 for further details). Panels B–D. Conservation of predicted and experimentally determined promoters across homologous regions from 18 Prochlorococcus marinus and Synechococcus spp. genomes. The regions were visualized using the WebLOGO web site (http://weblogo.berkeley.edu/). Panel B. Conservation of experimentally determined promoters from Prochlorococcus marinus MED4 [23]. Panel C. Conservation of predicted promoter located 37 bases into the trp-tRNA gene. Panel D. Conservation of the predicted promoter in the 5′ region 12–14 bases upstream of the PSHCP start codon. This promoter is present in all analyzed genomes except Synechococcus sp. RCC307. For promoters predicted upstream of the trp-tRNA and rpl19 genes (panel A) WebLOGOs are not shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215834&req=5

pone-0109327-g001: Schematic presentation of the genomic region around the PSHCP gene.Panel A. Mapping of promoters and primers onto the region. In all examined picocyanobacterial genomes the upstream region consists of the ribosomal protein L19 (rpL19) and tryptophanyl-tRNA genes. The locations of predicted promoters are shown by bent arrows, while the locations of the reverse (R1) and forward (F1 to F6) primers used for RT-Q-PCR analysis are indicated by half arrows (see Table S1 for further details). Panels B–D. Conservation of predicted and experimentally determined promoters across homologous regions from 18 Prochlorococcus marinus and Synechococcus spp. genomes. The regions were visualized using the WebLOGO web site (http://weblogo.berkeley.edu/). Panel B. Conservation of experimentally determined promoters from Prochlorococcus marinus MED4 [23]. Panel C. Conservation of predicted promoter located 37 bases into the trp-tRNA gene. Panel D. Conservation of the predicted promoter in the 5′ region 12–14 bases upstream of the PSHCP start codon. This promoter is present in all analyzed genomes except Synechococcus sp. RCC307. For promoters predicted upstream of the trp-tRNA and rpl19 genes (panel A) WebLOGOs are not shown.

Mentions: Cell pellets were obtained by centrifugation and RNA was extracted using the Trizol Kit (Life Technologies) as per the manufacturer's instructions. RNA extracts were treated with DNase I (Life Technologies) and quantified using the Quant-it RNA Broad Range Assay Kit (BR Kit, Life Technologies). Total RNA (60 ng) was reverse transcribed with random hexamer primers using the iScript cDNA Synthesis Kit (Bio-Rad), with reverse transcriptase omitted from the –RT negative control samples. Primers were designed to amplify specific regions surrounding the pshcp gene (Table S1 and Figure 1A). Calibrated standards were produced by amplifying the entire region of interest for all species (869 bp for WH 8102, 835 bp for MIT 9313, and 817 bp for MED4) using genomic DNA as a template. The PCR products were purified using a MinElute PCR Purification Kit (Qiagen) and quantified using the Quant-it DNA BR Kit (Life Technologies). Triplicate samples were prepared for RT-PCR using iQ SYBR Green Supermix (Bio-Rad) with a final cDNA content of 1.5 ng and 40 nM of forward and reverse primers. Samples were amplified using the following program in an iCycler thermal cycler (Bio-Rad): 95°C for 2 min; 40 cycles of 95°C for 30 s, 54°C for 30 s, 72°C for 30 s; 5 min at 72°C.


Quantitative and functional characterization of the hyper-conserved protein of Prochlorococcus and marine Synechococcus.

Whidden CE, DeZeeuw KG, Zorz JK, Joy AP, Barnett DA, Johnson MS, Zhaxybayeva O, Cockshutt AM - PLoS ONE (2014)

Schematic presentation of the genomic region around the PSHCP gene.Panel A. Mapping of promoters and primers onto the region. In all examined picocyanobacterial genomes the upstream region consists of the ribosomal protein L19 (rpL19) and tryptophanyl-tRNA genes. The locations of predicted promoters are shown by bent arrows, while the locations of the reverse (R1) and forward (F1 to F6) primers used for RT-Q-PCR analysis are indicated by half arrows (see Table S1 for further details). Panels B–D. Conservation of predicted and experimentally determined promoters across homologous regions from 18 Prochlorococcus marinus and Synechococcus spp. genomes. The regions were visualized using the WebLOGO web site (http://weblogo.berkeley.edu/). Panel B. Conservation of experimentally determined promoters from Prochlorococcus marinus MED4 [23]. Panel C. Conservation of predicted promoter located 37 bases into the trp-tRNA gene. Panel D. Conservation of the predicted promoter in the 5′ region 12–14 bases upstream of the PSHCP start codon. This promoter is present in all analyzed genomes except Synechococcus sp. RCC307. For promoters predicted upstream of the trp-tRNA and rpl19 genes (panel A) WebLOGOs are not shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215834&req=5

pone-0109327-g001: Schematic presentation of the genomic region around the PSHCP gene.Panel A. Mapping of promoters and primers onto the region. In all examined picocyanobacterial genomes the upstream region consists of the ribosomal protein L19 (rpL19) and tryptophanyl-tRNA genes. The locations of predicted promoters are shown by bent arrows, while the locations of the reverse (R1) and forward (F1 to F6) primers used for RT-Q-PCR analysis are indicated by half arrows (see Table S1 for further details). Panels B–D. Conservation of predicted and experimentally determined promoters across homologous regions from 18 Prochlorococcus marinus and Synechococcus spp. genomes. The regions were visualized using the WebLOGO web site (http://weblogo.berkeley.edu/). Panel B. Conservation of experimentally determined promoters from Prochlorococcus marinus MED4 [23]. Panel C. Conservation of predicted promoter located 37 bases into the trp-tRNA gene. Panel D. Conservation of the predicted promoter in the 5′ region 12–14 bases upstream of the PSHCP start codon. This promoter is present in all analyzed genomes except Synechococcus sp. RCC307. For promoters predicted upstream of the trp-tRNA and rpl19 genes (panel A) WebLOGOs are not shown.
Mentions: Cell pellets were obtained by centrifugation and RNA was extracted using the Trizol Kit (Life Technologies) as per the manufacturer's instructions. RNA extracts were treated with DNase I (Life Technologies) and quantified using the Quant-it RNA Broad Range Assay Kit (BR Kit, Life Technologies). Total RNA (60 ng) was reverse transcribed with random hexamer primers using the iScript cDNA Synthesis Kit (Bio-Rad), with reverse transcriptase omitted from the –RT negative control samples. Primers were designed to amplify specific regions surrounding the pshcp gene (Table S1 and Figure 1A). Calibrated standards were produced by amplifying the entire region of interest for all species (869 bp for WH 8102, 835 bp for MIT 9313, and 817 bp for MED4) using genomic DNA as a template. The PCR products were purified using a MinElute PCR Purification Kit (Qiagen) and quantified using the Quant-it DNA BR Kit (Life Technologies). Triplicate samples were prepared for RT-PCR using iQ SYBR Green Supermix (Bio-Rad) with a final cDNA content of 1.5 ng and 40 nM of forward and reverse primers. Samples were amplified using the following program in an iCycler thermal cycler (Bio-Rad): 95°C for 2 min; 40 cycles of 95°C for 30 s, 54°C for 30 s, 72°C for 30 s; 5 min at 72°C.

Bottom Line: Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production.The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments.We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Biochemistry, Mount Allison University, Sackville, NB, Canada.

ABSTRACT
A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

Show MeSH
Related in: MedlinePlus