Ice cover extent drives phytoplankton and bacterial community structure in a large north-temperate lake: implications for a warming climate.

Mid-winter limnological surveys of Lake Erie captured extremes in ice extent ranging from expansive ice cover in 2010 and 2011 to nearly ice-free waters in 2012. Consistent with a warming climate, ice cover on the Great Lakes is in decline, thus the ice-free condition encountered may foreshadow the lakes future winter state. Here, we show that pronounced changes in annual ice cover are accompanied by equally important shifts in phytoplankton and bacterial community structure. Expansive ice cover supported phytoplankton blooms of filamentous diatoms. By comparison, ice free conditions promoted the growth of smaller sized cells that attained lower total biomass. We propose that isothermal mixing and elevated turbidity in the absence of ice cover resulted in light limitation of the phytoplankton during winter. Additional insights into microbial community dynamics were gleaned from short 16S rRNA tag (Itag) Illumina sequencing. UniFrac analysis of Itag sequences showed clear separation of microbial communities related to presence or absence of ice cover. Whereas the ecological implications of the changing bacterial community are unclear at this time, it is likely that the observed shift from a phytoplankton community dominated by filamentous diatoms to smaller cells will have far reaching ecosystem effects including food web disruptions.

Diatom assemblages promote ice formation in large lakes.

We present evidence for the directed formation of ice by planktonic communities dominated by filamentous diatoms sampled from the ice-covered Laurentian Great Lakes. We hypothesize that ice formation promotes attachment of these non-motile phytoplankton to overlying ice, thereby maintaining a favorable position for the diatoms in the photic zone. However, it is unclear whether the diatoms themselves are responsible for ice nucleation. Scanning electron microscopy revealed associations of bacterial epiphytes with the dominant diatoms of the phytoplankton assemblage, and bacteria isolated from the phytoplankton showed elevated temperatures of crystallization (T(c)) as high as -3 °C. Ice nucleation-active bacteria were identified as belonging to the genus Pseudomonas, but we could not demonstrate that they were sufficiently abundant to incite the observed freezing. Regardless of the source of ice nucleation activity, the resulting production of frazil ice may provide a means for the diatoms to be recruited to the overlying lake ice, thereby increasing their fitness. Bacterial epiphytes are likewise expected to benefit from their association with the diatoms as recipients of organic carbon excreted by their hosts. This novel mechanism illuminates a previously undescribed stage of the life cycle of the meroplanktonic diatoms that bloom in Lake Erie and other Great Lakes during winter and offers a model relevant to aquatic ecosystems having seasonal ice cover around the world.

The unique proline of the Prochlorothrix hollandica plastocyanin hydrophobic patch impairs electron transfer to photosystem I.

A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.

Growth phase and metal-dependent regulation of the dpsA gene in Synechococcus sp. strain PCC 7942, USA.

The Synechococcus sp. strain PCC 7942 dpsA gene encodes a stress-inducible DNA-binding protein whose transcription increases in the stationary phase. Such transcription is likely under the control of an alternative sigma factor. Our current work indicated that dpsA transcription is also important under metal-ion limitation, because dpsA mRNA levels increased 12-fold under low-iron conditions, and that dpsA function is essential for growth under iron-limiting conditions. Promoter activity of the dpsA-promoter-lacZ reporter gene constructs implied that a region of dyad symmetry centered 28 nucleotides from the transcription start is required for metal-dependent repression, as judged by the level of lacZ induction following treatment of cultures with the chelator 2,2'-dipyridyl. This potential operator sequence is distinct from the site recognized by the cyanobacterial Fur repressor homologue. No other nutrient stresses (nitrogen, sulfur, phosphorus) yielded the high level of induction seen following chelator treatment. These studies suggest that there may be more than one class of metal-dependent repressor in cyanobacteria.

NMR solution structure of plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica.

The solution structure of a divergent plastocyanin (PC) from the photosynthetic prokaryote Prochlorothrix hollandica was determined by homonuclear 1H NMR spectroscopy. Nineteen structures were calculated from 1222 distance restraints, yielding a family of structures having an average rmsd of 0.42 +/- 0.08 A for backbone atoms and 0.71 +/- 0.07 A for heavy atoms to the mean structure. No distance constraint was violated by more than 0.26 A in the structure family. Despite the low number of conserved residues shared with other PC homologues, the overall folding pattern of P. hollandica PC is similar to other PCs, in that the protein forms a two-sheet beta-barrel tertiary structure. The greatest variability among the backbone structures is seen in the loop region from residues 47-60. The differences seen in the P. hollandica PC homologue likely arise due to a small deletion of 2-4 residues compared to the PC consensus; this yields a less extended loop containing a short alpha-helix from residues Ala52-Leu55. Additionally, the protein has an altered hydrophobic patch thought to be important in binding reaction partners. Whereas the backbone structure is very similar within the loops of the hydrophobic region, the presence of two unique residues (Tyr12 and Pro14) yields a structurally different hydrophobic surface likely important in binding P. hollandica Photosystem I.

Transcript analysis of the pcbABC genes encoding the antenna apoproteins in the photosynthetic prokaryote, Prochlorothrix hollandica.

The tightly linked pcbABC genes encode the chlorophyll a/b-binding apoproteins in the oxygenic photosynthetic prokaryote Prochlorothrix hollandica. Northern blotting experiments employing gene-specific DNA probes have identified a complex pattern of transcription from the pcb region. A large 4.4-kb transcript detected in cultures maintained in high light, low light and in darkness results from the cotranscription of all three genes, whereas pcbAB, pcbBC and individual pcbA, B, and C mRNAs are similarly detected in all light regimes. The half lives of the RNAs vary from 15 min for the pcbABC transcript, to over 60 min for the pcbA and pcbC mRNAs. The lack of identifiable promoter sequences other than the region upstream from pcbA, plus the enhanced stability of the individual single gene transcripts, suggest that the smaller RNA species arise from processing of larger transcripts. Transcription and mRNA turnover occurs largely independent of light intensity, in contrast to what is seen in most other phototrophs, in which light influences the accumulation of antenna apoprotein gene mRNAs.

Reconstitution and characterization of a divergent plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica, expressed in Escherichia coli.

Plastocyanin (PC) is a copper protein that serves as a mobile electron carrier between cytochrome f and Photosystem I in the light reactions of photosynthesis. Despite large variability in amino acid sequences and isoelectric points, PCs from cyanobacterial and chloroplast sources reveal considerable similarities with respect to their secondary and tertiary structures. In this paper, we have expressed in Escherichia coli a PC from the prokaryote Prochlorothrix hollandica, and efficiently reconstituted the protein with copper under conditions yielding the characteristics of a native holoPC, as judged by redox titration (Eo' = +376 mV), near and far UV circular dichroism, and electron paramagnetic resonance (EPR) spectroscopy. By comparison of amino acid sequences, P. hollandica PC is the most divergent homolog identified to date, and analysis of this reconstituted preparation may reveal new insights as to the structural requirements for electron transport between the PC copper center and neighboring reaction partners.

Expression of the petE gene encoding plastocyanin in the photosynthetic prokaryote, Prochlorothrix hollandica.

The expression of the petE gene encoding plastocyanin (PC) in the prokaryote Prochlorothrix hollandica is dependent on the presence of copper in the medium. PC protein and petE mRNA are detectable only under copper (Cu) replete conditions, suggesting that control of PC accumulation can occur at the level of transcription or transcript stability. Addition of Cu (0.3 microM) to log-phase Cu-deficient cultures yields accumulation of PC to detectable levels within 12 h; transfer of Cu-replete cells to Cu-deficient medium results in a slow decrease in the level of protein likely due to dilution by cell growth. By contrast, addition of high-affinity Cu-specific chelators to rapidly deplete Cu-grown cells of copper yields a rapid loss of PC with 2 h. These data suggest that Cu-free apoPC is turned over rapidly by proteolysis. Overall, these data demonstrate that regulation of PC levels as Cu levels change involve events occurring at the level of both RNA and protein turnover.

Cloning and functional analysis of the pmmA gene encoding phosphomannomutase from the photosynthetic prokaryote Prochlorothrix hollandica.

The pmmA gene encoding a bifunctional phosphomannomutase/phosphoglucomutase (PMM/PGM) from the photosynthetic prokaryote, Prochlorothrix hollandica has been cloned and sequenced. The gene encodes a 51827 Da polypeptide 48% identical to the PMM of Azospirillum brasilense, 37% identical to the PGMs of pathogenic Neisseria sp. and 37% identical to the bifunctional AlgC PGM/PMM of Pseudomonas aeruginosa. The pmmA gene encodes an enzyme having both PGM and PMM activities as judged by both enzyme assays and complementation analysis in which the cloned gene partially corrected the pgm-1 mutation of Escherichia coli.

The DpsA protein of Synechococcus sp. Strain PCC7942 is a DNA-binding hemoprotein. Linkage of the Dps and bacterioferritin protein families.

The Dps family of proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long term nutrient limitation. Some members of the Dps protein family have been shown to form large (approximately 150-kDa), hexameric complexes that bind chromosomal DNA with little sequence specificity. In this paper we report the nucleotide sequence of the dpsA gene from Synechococcus sp. PCC7942 encoding a cyanobacterial Dps homolog. The deduced amino acid sequence of the Synechococcus sp. DpsA protein revealed that a carboxyl-terminal domain of the protein was > 60% homologous to the COOH-terminal half of bacterioferritin. Other known Dps family members lack such high similarity to the bacterioferritins. Purification and spectroscopic analysis of the Synechococcus sp. DpsA protein complex revealed that the complex contains heme and has a weak catalase activity in vitro. Activity staining of nondenaturing polyacrylamide gels showed that the protein complex comigrated with both the heme and the catalase activity, and O2 evolution measurements yielded a maximal specific activity of 1.7 mumol of H2O2 consumed/micrograms of protein-1 min-1. We speculate that the protein may have a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggest that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Last, the evolutionary link between the Dps protein family and the bacterioferritins is discussed.

Purification and characterization of a Synechococcus sp. strain PCC 7942 polypeptide structurally similar to the stress-induced Dps/PexB protein of Escherichia coli.

A stable DNA/protein complex having an apparent molecular mass of approximately 150 kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.

Nucleotide sequence of the petE gene encoding plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica.

We have determined the nucleotide sequence of the petE gene encoding plastocyanin from the chlorophyll a/b-containing photosynthetic prokaryote, Prochlorothrix hollandica. Comparison of the deduced amino acid sequence encoded by the gene with the N-terminal sequence of the purified protein revealed that plastocyanin is synthesized as a precursor bearing an N-terminal domain of 34 amino acids having some structural similarity to thylakoid lumenal transit peptides identified in other organisms. The mature protein has an apparent isoelectric point of 8.37 and a molecular mass of 10,236 Da.

Light-Dependent Tyrosine Phosphorylation in the Cyanobacterium Prochlorothrix hollandica.

A light-dependent tyrosine kinase activity is present in soluble extracts from the cyanobacterium Prochlorothrix hollandica. The substrate of this tyrosine kinase activity is a soluble 88-kD protein that is phosphorylated when cultures of P. hollandica are adapted to high-light conditions. This phosphoprotein was identified by probing western blots of 32P-labeled soluble proteins from P. hollandica with an antibody specific for phosphotyrosine. This specificity was confirmed by competition experiments in which the antibody binding was abolished completely in the presence of excess phosphotyrosine but not phosphoserine and phosphothreonine. The kinetics of phosphorylation in vivo were determined by probing western blots with this antibody. Within 1 h following a switch from extended darkness to high light (200 [mu]mol photons m-2 s-1), the 88-kD protein was detectable upon India ink staining of western blots. After 3 h, the antibody recognized the phosphorylated form of this polypeptide. Within 6 h of a downshift from high to low light, the 88-kD protein was dephosphorylated. In vitro phosphorylation studies also showed that cell extracts can phosphorylate a tyrosine-containing artificial substrate; acid hydrolysis of both the artificial substrate and the 88-kD protein showed that phosphorylation occurred exclusively on tyrosine residues. Finally, experiments with high-light-adapted Synechococcus sp. PCC7942 suggest that a similar tyrosine phosphorylation event occurs in a phycobilisome-containing cyanobacterium.

The prochlorophytes: are they more than just chlorophyll a/b-containing cyanobacteria?

The prochlorophytes are a diverse group of photosynthetic prokaryotes that fall within the cyanobacterial lineage, yet lack phycobilisomes as light harvesting structures. Instead, the prochlorophytes have a light-harvesting apparatus composed of the higher plant pigments chlorophylls a and b. This review discusses the evolutionary relationships among these bacteria, with focus on the structure and function of the photosynthetic apparatus. This analysis yields a consensus from studies both on Prochloron sp. and Prochlorothrix hollandica as to how the thylakoid membrane is organized. Overall, we propose that the structure of the light-harvesting complexes (LHC) from prochlorophytes is very different from those of chloroplast systems, and is evolutionarily very ancient. The functional association of the light-harvesting apparatus with photosystem I (PSI) in both Prochlorothrix and Prochloron, as well as a demonstrated capacity for PSI-dependent anoxygenic photosynthesis in Prochlorothrix, may indicate that there is an increased dependence on cyclic photophosphorylation in these organisms. Finally, the structure of the prochlorophyte thylakoid membrane is discussed with respect to the forces that drive thylakoid membrane stacking in prochlorophytes and chloroplasts. We suggest that the light-harvesting structures in prochlorophytes play little, if any, role in this process.

Immunological characterization of the Prochlorothrix hollandica and Prochloron sp. chlorophyll a/b antenna proteins.

Polyclonal antibodies were prepared against the major antenna chlorophyll (Chl) a/b-binding protein from the prokaryote Prochlorothrix hollandica (Burger-Wiersma et al. (1986) Nature (Lond.) 320, 262-264). Immunoblotting experiments on Triton X-114 phase-partitioned P. hollandica thylakoids revealed that the antibody recognizes intrinsic membrane polypeptides of 33 and 30 kDa, and immunocytochemistry of P. hollandica thin sections showed that the antibody preferentially decorates the thylakoid. The antibody was immunopurified against a LacZ fusion protein produced in Escherichia coli by an immunopositive phage clone retrieved from a lambda ZAP expression library. This purified antibody crossreacted to both the 33 and 30 kDa polypeptides, indicating that these proteins are either structurally related products of different genes, or modified forms of the same gene product. Whereas immunological crossreactivity of Prochlorothrix antibody to the major LHC-II Chl a/b antenna of maize could not be detected, the immunopurified antibody reacted strongly to the major 34 kDa Chl a/b antenna protein from the prokaryote Prochloron sp. (Lewin (1975) Phycologia 14, 153-160). These data confirm the structural similarity of the prochlorophyte photosynthetic antenna systems.

Cloning, nucleotide sequence, and mutagenesis of a gene (irpA) involved in iron-deficient growth of the cyanobacterium Synechococcus sp. strain PCC7942.

We describe the cloning and sequencing of a gene from the cyanobacterium Synechococcus sp. strain PCC7942, designated irpA (iron-regulated protein A), that encodes for a protein involved in iron acquisition or storage. Polyclonal antibodies raised against proteins which accumulate during iron-deficient growth were used as probes to isolate immunopositive clones from a lambda gt11 genomic expression library. The clone, designated lambda gtAN26, carried a 1.7-kilobase (kb) chromosomal DNA insert and was detected by cross-reactivity with antibody against a 36-kilodalton protein. It was possible to map a 20-kb portion of the chromosome with various DNA probes from lambda gt11 and lambda EMBL-3 clones, and Southern blot analysis revealed that the irpA gene was present in a single copy and localized within a 1.7-kb PstI fragment. DNA sequencing revealed an open reading frame of 1,068 nucleotides capable of encoding 356 amino acids which yields a protein with a molecular weight of 38,584. The hydropathy profile of the polypeptide indicated a putative N-terminal signal sequence of 44 amino acid residues. IrpA is a cytoplasmic membrane protein as determined by biochemistry and electron microscopy immunocytochemistry. The upstream region of the irpA gene contained a consensus sequence similar to the aerobactin operator in Escherichia coli. This fact, plus a mutant with a mutation in irpA that is unable to grow under iron-deficient conditions, led us to suggest that irpA is regulated by iron and that the gene product is involved in iron acquisition or storage.

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b.

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.

Immunological characterization of photosystem II chlorophyll-binding proteins from the cyanobacterium, Aphanocapsa 6714.

Chlorophyll-binding proteins from the cyanobacterium Aphanocapsa 6714 were identified by immunoblotting procedures. Three chlorophyll-binding complexes, CPIII', CPIIIa, and CPIIIb, were associated with PSII. CPIII' likely serves as an' antenna to PSII in Aphanocapsa since it could be removed from active PSII core preparations without loss of activity. The CPIII' proteins cross-reacted to antibodies prepared against the maize PSII light-harvesting complex, LHC-II. The CPIIIa polypeptides cross-reacted to antibodies raised against the Chlamydomonas PSII chlorophyll-proteins 5 and 6, indicating that this complex contains the major chlorophyll-binding species of the cyanobacterial PSII core. Lastly, an antibody prepared against the canobacterial 36-kDa chlorophyll-binding protein [Pakrasi, H., Riethman, H., and Sherman, L. (1985). Proc. Natl. Acad. Sci. USA 82, 6903-6907] recognized only the 36-kDa IIIb apoprotein, indicating that CPIIIb represents a distinct chlorophyll-protein complex.

Identification of a carotenoid-binding protein in the cytoplasmic membrane from the heterotrophic cyanobacterium Synechocystis sp. strain PCC6714.

We isolated a carotenoid-binding protein from the cytoplasmic membrane of the cyanobacterium Synechocystis sp. strain PCC6714. The polypeptide demonstrated a characteristic mobility shift when electrophoresed in lithium dodecyl sulfate-polyacrylamide gels. The protein migrated with an apparent molecular mass of 35 kilodaltons when solubilized at 0 degrees C, but after solubilization at 70 degrees C, the protein migrated as a 45-kilodalton species. The carotenoid-binding protein accumulated only in autotrophically grown cells; cytoplasmic membranes prepared from photoheterotrophically grown cells lacked this component.