Ecology of purple sulfur bacteria in the highly stratified meromictic Lake Shunet (Siberia, Khakassia) in 2002–2009

Phototrophic sulfur bacteria form dense accumulations in the chemocline zones of stratified lakes where light reaches the sulfide-containing layers of water. Many works are dedicated to the ecophysiology of these microorganisms in meromictic lakes. However, the role of these microorganisms in the trophic network of these ecosystems, the ways of biomass utilization, and the contribution to the turnover of biogenic elements have so far been insufficiently understood. This work deals with the analysis of many years?? seasonal dynamics of the biomass of purple sulfur bacteria and the physicochemical conditions of their environment in Lake Shunet (Siberia, Khakassia, Russia), unraveling the causes of their anomalous development in the chemocline of this lake, as well as the comparative analysis of such type of ecosystems. Lake Shunet is characterized by markedly pronounced stratification and the high density of purple sulfur bacteria (PSB) in the chemocline, which is comparable to that of Lake Mahoney (Canada) where the number of PSB is the greatest among those known in the world. It was shown that, in the period 2002?C2009, the total amount of bacterio-chlorophyll a in the water column of Lake Shunet increased and did not correlate with the seasonal variations in temperature and illumination in the chemocline. It was established that PSB cells in the purple layer experienced the effect of self-shading. The sedimentation rate of purple sulfur bacteria in Lake Shunet was low due to the pronounced density gradient in the chemocline zone. Thus, the high number of PSB in the chemocline was due to the combination of strong illumination, a high sulfide concentration, and a high water density gradient, which was responsible for stable stratification and contributed to the accumulation of the cells in a narrow layer. The data obtained could be useful for the paleoreconstruction of climatically deter-mined changes in the level of the lake and its periods of meromixis by the presence of carotenoids and bacte-riochlorophylls in the bottom sediments.     

Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase ζ

To probe Pol ζ functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol ζ in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol ζ is 30% as active as wild-type Pol ζ when replicating undamaged DNA. L979F Pol ζ shares with wild-type Pol ζ the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol ζ is error-prone compared to wild-type Pol ζ, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol ζ in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol ζ also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol ζ, and perhaps wild-type Pol ζ, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo.     

Some generalizations based on stratification and vertical mixing in meromictic Lake Shira,Russia, in the period 2002–2009

In a brackish, temperate, 24-m-deep Lake Shira, the profiles of salinity, temperature, oxygen and sulfide concentrations were measured on a seasonal basis from 2002 to 2009. The lake was shown to be meromictic with autumnal overturn restricted to mixolimnion. The depth of mixolimnion and position of oxic–anoxic interface varied annually. The spring mixing processes contribute to the formation of mixolimnion in autumn. The exceptionally windy spring of 2007 caused the deepening of mixolimnion in the winter of 2008. The winter position of oxic–anoxic interface was affected by the position of lower boundary of mixolimnion in all winters. The salinity in the winter mixolimnion increased compared with the autumn because of freezing out of salts from the upper water layers meters during ice formation and their dissolution in water below. The profiles of salinity and temperature were simulated by the mathematical 1-D model of temperature and salinity conditions taking into account ice formation. The simulated profiles generally coincided with the measured ones. The coincidence implies that simplified one-dimensional model can be applied to roughly describe salinity and density profiles and mixing behavior of Lake Shira.     

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.     

Mutational hotspots in the TP53 gene and, possibly, other tumor suppressors evolve by positive selection

Background  

The mutation spectra of the TP53 gene and other tumor suppressors contain multiple hotspots, i.e., sites of non-random, frequent mutation in tumors and/or the germline. The origin of the hotspots remains unclear, the general view being that they represent highly mutable nucleotide contexts which likely reflect effects of different endogenous and exogenous factors shaping the mutation process in specific tissues. The origin of hotspots is of major importance because it has been suggested that mutable contexts could be used to infer mechanisms of mutagenesis contributing to tumorigenesis.     

The mutational spectra of gene p53 in different types of tumors]

The comparative analysis of the frequencies of nucleotide exchanges in mutational spectra of gene p53 (5-8 exons) between germline cancer-prone families (Li-Fraumeni syndrome), between somatic mutations in the tumors of different histogenesis and cell lines, obtained from them, was carried out. The nucleotide positions with high level of mutation events (mutational "hot spots"), typical for germline mutational spectra, tissue-specific patterns of their disappearing and appearing of new ones in solid tumors in vivo, nearly complete absence of "hot spots" in lymphomas and cell lines in vitro were revealed. The obtained results allowed to suggest, that one of the leading factor controlling hot-spots distribution in 5-8 exons of gene p53 is the specificity of cell division conditions in vivo and in vitro.     

Microbial metabolism of the carbon and sulfur cycles in Shira Lake (Khakasia)

Microbiological and biogeochemical studies of the meromictic saline Lake Shira (Khakasia) were conducted. In the upper part of the hydrogen-sulfide zone, at a depth of 13.5-14 m, there was a pale pink layer of water due to the development of purple bacteria (6 x 10(5) cells/ml), which were assigned by their morphological and spectral characteristics to Lamprocystis purpureus (formerly Amoebobacter purpurea). In August, the production of organic matter (OM) in Lake Shira was estimated to be 943 mg C/(m2 day). The contribution of anoxygenic photosynthesis was insignificant (about 7% of the total OM production). The share of bacterial chemosynthesis was still less (no more than 2%). In the anaerobic zone, the community of sulfate-reducing bacteria played a decisive role in the terminal decomposition of OM. The maximal rates of sulfate reduction were observed in the near-bottom water (114 micrograms S/(1 day)) and in the surface layer of bottom sediments (901 micrograms S/(dm3 day)). The daily expenditure of Corg for sulfate reduction was 73% of Corg formed daily in the processes of oxygenic and anoxygenic photosynthesis and bacterial chemosynthesis. The profile of methane distribution in the water column and bottom sediments was typical of meromictic reservoirs. The methane content in the water column increased beginning with the thermocline (7-8 m), and reached maximum values in the near-bottom water (17 microliters/l). In bottom sediments, the greatest methane concentrations (57 microliters/l) were observed in the surface layer (0-3 cm). The integral rate of methane formation in the water column and bottom sediments was almost an order of magnitude higher than the rate of its oxidation by aerobic and anaerobic methanotrophic microorganisms.