George S. Bullerjahn

Ecophysiological Characterization of Ammonia-Oxidizing Archaea and Bacteria from Freshwater

ABSTRACT Aerobic biological ammonia oxidation is carried out by two groups of microorganisms, ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). Here we present a study using cultivation-based methods to investigate the differences in growth of three AOA cultures and one AOB culture enriched from freshwater environments. The strain in the enriched AOA culture belong to thaumarchaeal group I.1a, with the strain in one enrichment culture having the highest identity with “Candidatus Nitrosoarchaeum koreensis” and the strains in the other two representing a new genus of AOA. The AOB strain in the enrichment culture was also obtained from freshwater and had the highest identity to AOB from the Nitrosomonas oligotropha group (Nitrosomonas cluster 6a). We investigated the influence of ammonium, oxygen, pH, and light on the growth of AOA and AOB. The growth rates of the AOB increased with increasing ammonium concentrations, while the growth rates of the AOA decreased slightly. Increasing oxygen concentrations led to an increase in the growth rate of the AOB, while the growth rates of AOA were almost oxygen insensitive. Light exposure (white and blue wavelengths) inhibited the growth of AOA completely, and the AOA did not recover when transferred to the dark. AOB were also inhibited by blue light; however, growth recovered immediately after transfer to the dark. Our results show that the tested AOB have a competitive advantage over the tested AOA under most conditions investigated. Further experiments will elucidate the niches of AOA and AOB in more detail.

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 (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 O evolution measurements yielded a maximal specific activity of 1.7 μmol of HO consumed/μg of protein min. 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.

Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria

Microbial rhodopsins are membrane proteins that utilize a retinal chromophore to harvest sunlight for energetic and photosensory functions. Recently, a group of novel rhodopsin sequences named ‘actinorhodopsins’ (ActRs) was hypothesized to exist among uncultured planktonic Actinobacteria. ActRs were discovered by mining metagenomic data obtained during the Venter Institutes Global Ocean Sampling expedition, from a hypersaline lagoon, two estuaries and a freshwater lake. On the basis of these findings, and many studies that show Actinobacteria are common inhabitants of lakes, we predicted that ActR genes would likely be present in other freshwater habitats and among the genomes of cultivated Actinobacteria. Using degenerate polymerase chain reaction primers, we discovered an ActR gene present in an actinobacterial isolate of the family Microbacteriaceae. Isolate MWH-Uga1 was cultivated prior to this study from a freshwater pond in Uganda and belongs to a group of Actinobacteria previously identified in freshwater ecosystems. ActR genes were also discovered present in numerous mixed cultures containing freshwater Actinobacteria and among environmental DNA samples obtained from three freshwater sources; a small woodland pond and the Laurentian Great Lakes Superior and Erie. An analysis of small subunit ribosomal RNA genes from metagenomic DNA samples harboring ActR genes suggests that organisms belonging to the acI lineage, an uncultured group of Actinobacteria commonly present in fresh waters, may utilize rhodopsins. The co-occurrence of an acI organism with a specific ActR variant in a mixed culture supports our hypothesis.

Increasing stoichiometric imbalance in North America's largest lake: Nitrification in Lake Superior

more than 600 times the mean requirement ratio for primary producers. We examine the rate of [NO3 ] increase relative to budgets for NO3 and fixed N. Nitrate in Lake Superior has continued to rise since 1980, though possibly at a reduced rate. We constructed whole-lake NO3 and N budgets and found that NO3 must be generated in the lake at significant rates. Stable O isotope results indicate that most NO3 in the lake originated by in-lake oxidation. Nitrate in the lake is responding not just to NO3 loading but also to oxidation of reduced forms of nitrogen delivered to the lake. The increasing [NO 3 ]:[PO4 ]s toichiometric imbalance in this large lake is largely determined by these in-situ processes. Citation: Sterner, R. W., E. Anagnostou, S. Brovold, G. S. Bullerjahn, J. C. Finlay, S. Kumar, R. M. L. McKay, and R. M. Sherrell (2007), Increasing stoichiometric imbalance in North America’s largest lake: Nitrification in Lake Superior, Geophys. Res. Lett., 34, L10406, doi:10.1029/ 2006GL028861.

Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton

ABSTRACT Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the worlds lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 105 cells ml−1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.

Detection and expression of the phosphonate transporter gene phnD in marine and freshwater picocyanobacteria

We describe a PCR-based assay designed to detect expression of the phosphonate assimilation gene phnD from picocyanobacteria. The phnD gene encodes the phosphonate binding protein of the ABC-type phosphonate transporter, present in many of the picocyanobacterial genome sequences. Detection of phnD expression can indicate a capacity of picoplankton to utilize phosphonates, a refractory form of phosphorus that can represent 25% of the high-molecular-weight dissolved organic phosphorus pool in marine systems. Primer sets were designed to specifically amplify phnD sequences from marine and freshwater Synechococcus spp., Prochlorococcus spp. and environmental samples from the ocean and Laurentian Great Lakes. Quantitative RT-PCR from cultured marine Synechococcus sp. strain WH8102 and freshwater Synechococcus sp. ARC-21 demonstrated induction of phnD expression in P-deficient media, suggesting that phn genes are regulated coordinately with genes under phoRB control. Last, RT-PCR of environmental RNA samples from the Sargasso Sea and Pacific Ocean detected phnD expression from the endemic picocyanobacterial population. Synechococcus spp. phnD expression yielded a depth-dependent pattern following gradients of P bioavailability. By contrast, the Prochlorococcus spp. primers revealed that in all samples tested, phnD expression was constitutive. The method described herein will allow future studies aimed at understanding the utilization of naturally occurring phoshonates in the ocean as well as monitoring the acquisition of synthetic phosphonate herbicides (e.g. glyphosate) by picocyanobacteria in freshwaters.

Effects of Increasing Nitrogen and Phosphorus Concentrations on Phytoplankton Community Growth and Toxicity During Planktothrix Blooms in Sandusky Bay, Lake Erie

Sandusky Bay experiences annual toxic cyanobacterial blooms dominated by Planktothrix agardhii/suspensa. To further understand the environmental drivers of these events, we evaluated changes in the growth response and toxicity of the Planktothrix-dominated blooms to nutrient amendments with orthophosphate (PO4) and inorganic and organic forms of dissolved nitrogen (N; ammonium (NH4), nitrate (NO3) and urea) over the bloom season (June - October). We complemented these with a metagenomic analysis of the planktonic microbial community. Our results showed that bloom growth and microcystin (MC) concentrations responded more frequently to additions of dissolved N than PO4, and that the dual addition of NH4 + PO4 and Urea + PO4 yielded the highest MC concentrations in 54% of experiments. Metagenomic analysis confirmed that P. agardhii/suspensa was the primary MC producer. The phylogenetic distribution of nifH revealed that both heterocystous cyanobacteria and heterotrophic proteobacteria had the genetic potential for N2 fixation in Sandusky Bay. These results suggest that as best management practices are developed for P reductions in Sandusky Bay, managers must be aware of the negative implications of not managing N loading into this system as N may significantly impact cyanobacterial bloom size and toxicity.

Seasonal Hypoxia and the Genetic Diversity of Prokaryote Populations in the Central Basin Hypolimnion of Lake Erie: Evidence for Abundant Cyanobacteria and Photosynthesis

ABSTRACT The reoccurring region of seasonal hypoxia in the central basin of Lake Erie (“the dead zone”) has been of significant interest to researchers over the past several years. Surprisingly however, no efforts to characterize the endemic microbial community, responsible for the consumption of oxygen in this system, have been published. To understand how the microbial community may be interacting with this event, we have begun to characterize microbial members by using molecular tools. Phycoerythrin-rich cyanobacteria appear abundant and active in a narrow region (∼ 1.5 m) below the thermocline during hypoxic conditions, reaching abundances of greater than 105mL−1and being the primary agent releasing 1.5 mg O2 L−1above the daytime demands in this region. Sequencing of 16S rDNA amplicons, generated with universal eubacterial primer sets, from the Lake Eries hypolimnion during seasonal oxygen depletion demonstrated that cyanobacteria, most closely related to phycoerythrin-rich Synechococcus spp., dominate during rapid drawdown of oxygen (0.083 mg L−1d−1in 2004) in this region. Analyses of another conserved marker of phylogeny (RuBisCO) has been used to confirm the presence of these cell types. Numerous distinct taxa of heterotrophic bacteria are also represented in the 16S library. The results of this study suggest that novel groups of cyanobacteria may persist within the Lake Erie dead zone during hypoxic conditions and, along with the heterotrophic community, strongly influence system geochemistry.

Dynamics In The Transient Complex Of Plastocyanin-cytochrome F From Prochlorothrix Hollandica

The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between plastocyanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc-cytf complex. The orientation of Pc is similar to orientations found in Pc-cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wild-type Pc and mutant Pc, but the chemical shift perturbations are smaller for the complex with mutant Pc. Docking calculations for the Y12G/P14L Pc-cytf complex did not produce a converged ensemble of structures. Simulations of the dynamics were performed using the observed averaged NMR parameters as input. The results indicate a surprisingly large amplitude of mobility of Y12G/P14L Pc within the complex. It is concluded that the double mutation shifts the complex further from the well-defined toward the encounter state.

Luminescent whole-cell cyanobacterial bioreporter for measuring Fe availability in diverse marine environments.

ABSTRACT A Synechococcus sp. strain PCC 7002 Fe bioreporter was constructed containing the isiAB promoter fused to the Vibrio harveyi luxAB genes. Bioreporter luminescence was characterized with respect to the free ferric ion concentration in trace metal-buffered synthetic medium. The applicability of the Fe bioreporter to assess Fe availability in the natural environment was tested by using samples collected from the Baltic Sea and from the high-nutrient, low-chlorophyll subarctic Pacific Ocean. Parallel assessment of dissolved Fe and bioreporter response confirmed that direct chemical measurements of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton.