Ora Hadas

Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment

The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments.

A Synechococcus PglnA::luxAB Fusion for Estimation of Nitrogen Bioavailability to Freshwater Cyanobacteria

ABSTRACT In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 μM for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.

Evaluation of PCR Primer Selectivity and Phylogenetic Specificity by Using Amplification of 16S rRNA Genes from Betaproteobacterial Ammonia-Oxidizing Bacteria in Environmental Samples†

ABSTRACT The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (βAOB) was evaluated. βAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the βAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations βAMOf/βAMOr, βAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on βAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

Effect of salinity on cyanobacterial community composition along a transect from Fuliya spring into the water of Lake Kinneret, Israel

Cyanobacterial community composition was studied along a salinity gradient from the saline Spring Fuliya towards the water column of Lake Kinneret. The samples included a gradient of salinities ranging from 4270 mg Cl L-1 (Saline Spring) to 239 mg Cl L-1 (Lake Kinneret). Denaturing gradient gel electrophoresis (DGGE) and cloning of the 16 S rRNA gene, as well as cloning and sequencing of the psbA gene, were used to characterize cyanobacterial community composition. Despite the differences in salinity, similar cyanobacterial communities were observed in the lake and the saline spring, the only exception being the highest salinity sample (4270 mg Cl L-1). Both, DGGE patterns and results of the clone libraries revealed the dominance of cyanobacteria with colonial Gloeocapsa and unicellular Synechococcus as the closest known cultured relatives, independently of the salinity. These results suggest that cyanobacterial populations inhabiting this freshwater lake and its saline sources can adapt to a wide range of salinities.

The Fate of Organic Carbon

In Lake Kinneret, the majority of photosyntetically produced organic carbon (OC) is cycled through the microbial loop. Taken together, bacterial production (BP) and bacterial respiration (BR), i.e., bacterial carbon demand (BCD), accounted for about 65 % of gross primary production (GPP), measured biweekly and averaging 2.3 g C m–2 day–1 during the last decade (2001–2011). Community respiration (CR) was 2.1 g C m–2 day–1. The major contributors to total CR were bacterial and phytoplankton respiration (~80%) while zooplankton respiration accounted for the reminder. Most (~ 83 %) of the OC input were eventually respired, ~3 % lost to outflows, while ~15 % of the total OC input were transferred annually to the sediments. Here oxic mineralization is gradually replaced by anoxic processes as a function of the availability of suitable electron acceptors. After the depletion of oxygen in the hypolimnion, sulfate (500 μM) becomes the dominant oxidant. Depending on the settling flux of OC sedimentary sulfate reduction (SR) rates were measured from 0.01 to 1.67 µmol cm–3 day–1 in December and July, respectively. SR is the dominant anaerobic terminal decomposition process in Lake Kinneret and is responsible for the accumulation of sulfide in the hypolimnion to concentrations up to 400 μM. Methanogenesis is restricted to those sediment layers that are depleted of sulfate (below 3–5 cm). Seasonal profiles and 13C signatures of dissolved methane in the sediment pore water of Lake Kinneret have indicated anaerobic methane oxidation in the deeper sediments (below 20 cm), with Fe(III) as electron acceptor. Lake Kinneret resembles the first aquatic ecosystem where the existence of this process could be verified. Changes in the watershed and lake environment are suggested as possible causes for the apparently significant declines in bacterial numbers, BP, and BCD that have taken place over the last decade in Lake Kinneret.

Protozoa (Unicellular Zooplankton): Ciliates and Flagellates

Heterotrophic and mixotrophic protozoa have been recognized as important constituents of aquatic microbial food webs since the 1990s. Much less is known about the ecosystem roles of these organisms than about bacteria and archaea. Several pioneering studies in Lake Kinneret made in the 1980s clearly indicated that heterotrophic and mixotrophic ciliates and flagellates were of great significance in the mineralization of organic matter and cycling of carbon, nitrogen, and phosphorus. Modeling studies suggested that the protozoa, especially ciliates, appeared to be a critical food source for copepods. However, only after many years there was renewed research on protozoa as drivers of carbon flux and nutrient cycling. Routine monitoring of the lake ciliate populations was begun in 2006.