Vladimir Yurkov

Isolation of Tellurite- and Selenite-Resistant Bacteria from Hydrothermal Vents of the Juan de Fuca Ridge in the Pacific Ocean

ABSTRACT Deep-ocean hydrothermal-vent environments are rich in heavy metals and metalloids and present excellent sites for the isolation of metal-resistant microorganisms. Both metalloid-oxide-resistant and metalloid-oxide-reducing bacteria were found. Tellurite- and selenite-reducing strains were isolated in high numbers from ocean water near hydrothermal vents, bacterial films, and sulfide-rich rocks. Growth of these isolates in media containing K2TeO3 or Na2SeO3 resulted in the accumulation of metallic tellurium or selenium. The MIC of K2TeO3 ranged from 1,500 to greater than 2,500 μg/ml, and the MIC of Na2SeO3 ranged from 6,000 to greater than 7,000 μg/ml for 10 strains. Phylogenetic analysis of 4 of these 10 strains revealed that they form a branch closely related to members of the genus Pseudoalteromonas, within the γ-3 subclass of the Proteobacteria. All 10 strains were found to be salt tolerant, pH tolerant, and thermotolerant. The metalloid resistance and morphological, physiological, and phylogenetic characteristics of newly isolated strains are described.

New Light on Aerobic Anoxygenic Phototrophs

Discovered 30 years ago, aerobic anoxygenic phototrophs (AAP) represent an entirely new bacterial functional group that was surprisingly found to constitute nearly 10% of microbial cells in the world’s biggest surface ecosystem, the ocean. These intriguing and colorful descendents of anaerobic anoxygenic phototrophs possess a fully functional photosynthetic apparatus that is paradoxically operative only under oxic conditions. An obviously ancient group, the AAP display numerous extensive evolutionary modifications to their photosynthetic machinery from that of their ancestors, such as different suites of light-harvesting 2 complexes and, in some species, the only zinc-based chlorophyll pigments found anywhere in nature. Whereas AAP are incapable of photoautotrophy and rely on heterotrophy for 80% or more of their cellular energetics, sunlight can double organic carbon assimilatory efficiency over that of strict heterotrophs, making AAP key players in the marine carbon cycle. The AAP inhabit not just soil, rivers and oceans, but also hypersaline waters, thermal springs and even the dark realm of deep ocean hydrothermal vents. Ubiquity and atypical photosynthetic nature has inspired an ever-increasing scientific interest in the AAP, for which there are more exceptions than rules.

Aerobic Phototrophic Bacteria: New Evidence for the Diversity, Ecological Importance and Applied Potential of this Previously Overlooked Group

The aerobic phototrophic bacteria are a recently discovered group capable of producing a photosynthetic apparatus similar to that of purple phototrophic bacteria. However, this apparatus, in contrast to that of their anaerobic counterparts, is functional in terms of photoinduced electron transport only under aerobic conditions. Although these bacteria have been widely studied, little is yet known about their ecological importance, and why they differ from other anoxygenic phototrophs with respect to oxygen requirements. In recent years a large number of new genera and species have been described from a wide variety of habitats, and evidence has been presented to support their important ecological role. This minireview focuses on recent discoveries regarding taxonomy, ecology and physiology, as well as the latest advances in the understanding of their photosynthetic apparatus and its genetic regulation.

Diversity, distribution and physiology of the aerobic phototrophic bacteria in the mixolimnion of a meromictic lake

The population of anoxygenic phototrophic bacteria in the aerobic zone of the meromictic Mahoney Lake was investigated using classical microbiological methods. This bacterial community was found to be very rich and diverse. Thirty-one new strains of the obligately aerobic phototrophic bacteria, and two new purple nonsulfur strains, were isolated in pure cultures and preliminarily characterized. The isolates contain a variety of carotenoids, bacteriochlorophyll a incorporated into pigment protein complexes, and are morphologically and physiologically diverse. These properties indicate a diversity of adaptations to the stratified environments of this meromictic lake. Phylogenetically all isolated strains belong to the alpha subclass of Proteobacteria.

Diverse Arrangement of Photosynthetic Gene Clusters in Aerobic Anoxygenic Phototrophic Bacteria

Background Aerobic anoxygenic photototrophic (AAP) bacteria represent an important group of marine microorganisms inhabiting the euphotic zone of the ocean. They harvest light using bacteriochlorophyll (BChl) a and are thought to be important players in carbon cycling in the ocean. Methodology/Principal Findings Aerobic anoxygenic phototrophic (AAP) bacteria represent an important part of marine microbial communities. Their photosynthetic apparatus is encoded by a number of genes organized in a so-called photosynthetic gene cluster (PGC). In this study, the organization of PGCs was analyzed in ten AAP species belonging to the orders Rhodobacterales, Sphingomonadales and the NOR5/OM60 clade. Sphingomonadales contained comparatively smaller PGCs with an approximately size of 39 kb whereas the average size of PGCs in Rhodobacterales and NOR5/OM60 clade was about 45 kb. The distribution of four arrangements, based on the permutation and combination of the two conserved regions bchFNBHLM-LhaA-puhABC and crtF-bchCXYZ, does not correspond to the phylogenetic affiliation of individual AAP bacterial species. While PGCs of all analyzed species contained the same set of genes for bacteriochlorophyll synthesis and assembly of photosynthetic centers, they differed largely in the carotenoid biosynthetic genes. Spheroidenone, spirilloxanthin, and zeaxanthin biosynthetic pathways were found in each clade respectively. All of the carotenoid biosynthetic genes were found in the PGCs of Rhodobacterales, however Sphingomonadales and NOR5/OM60 strains contained some of the carotenoid biosynthetic pathway genes outside of the PGC. Conclusions/Significance Our investigations shed light on the evolution and functional implications in PGCs of marine aerobic anoxygenic phototrophs, and support the notion that AAP are a heterogenous physiological group phylogenetically scattered among Proteobacteria.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na+/Ca2+ exchangers (NCX) constitute a major Ca2+ export system that facilitates the re-establishment of cytosolic Ca2+ levels in many tissues. Ca2+ interactions at its Ca2+ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na+/Ca2+ exchange activity. The structure of the Ca2+-bound form of CBD1, the primary Ca2+ sensor from canine NCX1, but not the Ca2+-free form, has been reported, although the molecular mechanism of Ca2+ regulation remains unclear. Here, we report crystal structures for three distinct Ca2+ binding states of CBD1 from CALX, a Na+/Ca2+ exchanger found in Drosophila sensory neurons. The fully Ca2+-bound CALX-CBD1 structure shows that four Ca2+ atoms bind at identical Ca2+ binding sites as those found in NCX1 and that the partial Ca2+ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca2+ binding at CBD1. The structures also predict that the primary Ca2+ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu455, which coordinates the primary Ca2+ pair, produces dramatic reductions of the regulatory Ca2+ affinity for exchange current, whereas mutagenesis of Glu520, which coordinates the secondary Ca2+ pair, has much smaller effects. Furthermore, our structures indicate that Ca2+ binding only enhances the stability of the Ca2+ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca2+ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

A new environment for aerobic anoxygenic phototrophic bacteria: biological soil crusts

Phototrophic microorganisms are critical to the carbon cycling and productivity of biological soil crusts, which enhance water content, nutrient relations and mechanical stability of arid soils. Only oxygen-producing phototrophs, including cyanobacteria and algae, are known from soil crusts, but Earths second major branch of photosynthetic organisms, the evolutionarily earlier anoxygenic phototrophs, is unreported. We announce the discovery of aerobic anoxygenic phototrophs in three Canadian soil crust communities. We found in a culture-based study that they comprised 0.1-5.9% of the cultivable bacterial community in moss-, lichen- and cyanobacteria-dominated crust from sand dunes and sandy soils. Comparable in density to aerobic phototrophs in other habitats, the bacteriochlorophyll a-possessing pink and orange isolates were related to species of Methylobacterium (99.0-99.5%), Belnapia (97.4-98.8%), Muricoccus (94.4%) and Sphingomonas (96.6-98.5%), based on 16S rRNA gene sequences. Our results demonstrate that proteobacterial anoxygenic phototrophs may be found in dry soil environments, implying desiccation resistance as yet unreported for this group. By utilizing sunlight for part of their energy needs, aerobic phototrophs can accelerate organic carbon cycling in nutrient-poor arid soils. Their effects will be especially important as global climate change enhances soil erosion and consequent nutrient loss.

The major part of polar carotenoids of the aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 is not bound to the bacteriochlorophyll a-complexes of the photosynthetic apparatus

The obligate aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 contain numerous polar carotenoids. The major carotenoid of the strain RB3 was the C30 carotene-dioate (4,4′-diapocarotene-4,4′-dioate) and the respective diglycosyl ester which have never been isolated before from a bacteriochlorophyll containing bacterium. Strain E5 contains the very polar erythroxanthin sulphate. The major carotenoid bound to reaction center and light-harvesting complexes is bacteriorubixanthinal. Most of the carotenoids of both strains are not bound to the pigment-protein complexes of the photosynthetic apparatus but to the envelope fraction (cytoplasmic membrane and cell wall).

A puhA gene deletion and plasmid complementation system for facile site directed mutagenesis studies of the reaction center H protein of Rhodobacter sphaeroides

We describe the development of a Rhodobacter sphaeroides RC H (puhA) gene deletion/plasmid complementation system for expression of site directed mutants of the RC H protein. The mutant strain ΔPUHA was constructed by introduction of a translationally in-frame deleted puhA allele at the chromosomal puhA gene site, and evaluated in plasmid complementation experiments. Strain ΔPUHA was not capable of photosynthetic growth, and SDS-PAGE chromatophore proteins confirmed the absence of the RC H protein band. When ΔPUHA was complemented with the wild type puhA gene in plasmids, photosynthetic growth and the RC H protein band in SDS-PAGE were restored. The results of comparisons of the properties of strains with different types of chromosomal puhA gene disruptions in complementation experiments with plasmid-borne DNA fragments containing the wild type puhA gene are consistent with the idea that expression of one or more genes located 3′ of puhA is required for optimal RC levels and photosynthetic growth. Since the ΔPUHA translationally in frame deletion does not seem to interfere with transcription through and beyond the residual puhA sequences, this strain allows facile evaluation of the consequences of plasmid-borne RC H mutations in an otherwise wild type genetic background. Two plasmid-borne site directed mutants of the puhA gene (GluH173 → Gln and GluH173 → Asp) exhibited different deficiencies in photosynthetic growth when present in ΔPUHA, indicating that a carboxylic acid amino acid side chain at the H173 position is important for RC function.

Porphyrobacter meromictius sp. nov., an appendaged bacterium, that produces Bacteriochlorophyll a.

Four Gram-negative strains (ML4T, ML19, ML31, ML32) of nonmotile, appendaged, budding bacteria were isolated from the meromictic Mahoney Lake in British Columbia, Canada. The strains were red to brown-red in color and produced bacteriochlorophyll a incorporated into photosynthetic pigment-protein complexes. Phylogenetic analysis has placed these strains within the class Alphaproteobacteria, with the closest relatives being members of the genera Erythrobacter, Porphyrobacter, and Erythromicrobium. Morphological features warrant their inclusion within the genus Porphyrobacter and these strains can be readily distinguished from other species of this genus on the basis of a mesophilic temperature range, a broad pH range, and tolerance to extremely high NaCl and Na2SO4 concentrations, in keeping with the environment from which they were isolated, a Na2SO4-dominated meromictic lake. These isolates utilize a variety of organic substrates for aerobic chemoheterotrophic growth and do not grow under anaerobic conditions, in either the presence or the absence of light. All strains require vitamin B12, and strains ML4T and ML19 require biotin. The DNA G + C contents ranged from 62.2 to 64.9 mol%. Phenotypic and phyletic data support the classification of strains ML4T, ML19, ML31, and ML32 as a novel Porphyrobacter species for which the name Porphyrobacter meromictius sp. nov. is proposed.