Danny Ionescu

Selective enrichment, isolation and molecular detection of Salinibacter and related extremely halophilic Bacteria from hypersaline environments

Salinibacter is a genus of red, extremely halophilic Bacteria. Thus far the genus is represented by a single species, Salinibacter ruber, strains of which have been isolated from saltern crystallizer ponds in Spain and on the Balearic Islands. Both with respect to its growth conditions and its physiology, Salinibacter resembles the halophilic Archaea of the order Halobacteriales. We have designed selective enrichment and isolation techniques to obtain Salinibacter and related red extremely halophilic Bacteria from different hypersaline environments, based on their resistance to anisomycin and bacitracin, two antibiotics that are potent inhibitors of the halophilic Archaea. Using direct plating on media containing bacitracin, we found Salinibacter-like organisms in numbers between 1.4×103 and 1.4×106ml−1 in brines collected from the crystallizer ponds of the salterns in Eilat, Israel, being equivalent to 1.8–18% of the total colony counts obtained on identical media without bacitracin. A number of strains from Eilat were subjected to a preliminary characterization, and they proved similar to the type strain of S. ruber. We also report here the isolation and molecular detection of Salinibacter-like organisms from an evaporite crust on the bottom of salt pools at the Badwater site in Death Valley, CA. These isolates and environmental 16S rRNA gene sequences differ in a number of properties from S. ruber, and they may represent a new species of Salinibacter or a new related genus.

Heterocyst-specific transcription of NsiR1, a non-coding RNA encoded in a tandem array of direct repeats in cyanobacteria.

In response to nitrogen deficiency, some cyanobacteria develop heterocysts, a terminally differentiated cell type, specialized for the fixation of atmospheric nitrogen. In Nostocales, this differentiation process is controlled by two major regulators, NtcA and HetR, but additional unknown factors are likely to be involved as well. In the context of a genome-wide search for potential non-coding RNAs, we identified an array of 12 tandem repeats that is transcribed in large amounts when cells enter conditions that trigger cell differentiation and switch to nitrogen fixation. The main accumulating transcript, which we suggest designating nitrogen stress-induced RNA 1 (NsiR1), has properties similar to regulatory non-coding RNAs. In Anabaena sp. PCC 7120, it is about 60 nt in length, has a very distinct predicted secondary structure, and is expressed very early and transiently after nitrogen step-down. Moreover, its expression requires HetR and NtcA and is restricted to cells that are differentiating into heterocysts, clearly placing NsiR1 within the regulon that controls the switch to nitrogen fixation and heterocyst formation. The genomic arrangement of NsiR1, located upstream of hetF, a gene whose product is involved in heterocyst formation, is conserved in all five Nostocales whose genomes are completely sequenced. Additionally, we detected NsiR1 expression in 19 different heterocyst-forming cyanobacteria. Our data suggest that every repeat is a complete transcriptional unit furnished with a cell-type-specific promoter and a Rho-independent terminator, which gives rise to a very high NsiR1 transcript level. NsiR1 is the first known bacterial non-coding RNA that is specifically upregulated in response to nitrogen step-down.

Archaea in the Gulf of Aqaba

Using a polyphasic approach, we examined the presence of Archaea in the Gulf of Aqaba, a warm marine ecosystem, isolated from major ocean currents and subject to pronounced seasonal changes in hydrography. Catalyzed reported deposition FISH analyses showed that Archaea make up to >20% of the prokaryotic community in the Gulf. A spatial separation between the two major phyla of Archaea was observed during summer stratification. Euryarchaeota were found exclusively in the upper 200 m, whereas Crenarchaeota were present in greater numbers in layers below the summer thermocline. 16S rRNA gene-based denaturing gradient gel electrophoresis confirmed this depth partitioning and revealed further diversity of Crenarchaeota and Euryarchaeota populations along depth profiles. Phylogenetic analysis showed pelagic Crenarchaeota and Euryarchaeota to differ from coral-associated Archaea from the Gulf, forming distinct clusters within the Marine Archaea Groups I and II. Endsequencing of fosmid libraries of environmental DNA provided a tentative identification of some members of the archaeal community and their role in the microbial community of the Gulf. Incorporation studies of radiolabeled leucine and bicarbonate in the presence of different inhibitors suggest that the archaeal community participates in autotrophic CO(2) uptake and contributes little to the heterotrophic activity.