Generation of dormant forms by <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> in the poststationary phase during gradual acidification of the medium

Persistent forms of the wild-type strain of Mycobacterium smegmatis and its mutants with inactivated devR and hlp genes were investigated. devR encodes the regulatory protein responsible for the formation of nonreplicating mycobacterial forms under hypoxia, and hlp codes for a histone-like protein. It has been found that a gradual decrease of pH in M. smegmatis wild-type poststationary cultures resulted in the formation of a special type of persisters. They significantly differed from vegetative cells in their properties and were represented by shortened ovoid forms with thickened cell walls. According to atomic force microscopy data, the size of the ovoid forms and vegetative cells was 1.2 × 0.9 μm and 3.7 × 0.8 μm, respectively. The metabolism level was markedly decreased in ovoid cells: the incorporation of [5,6-³H]uracil and thymidine was decreased 200- and 50-fold, respectively. The intracellular ATP content was lowered threefold. The ovoid forms that emerged in poststationary cultures in Sauton’s medium when the medium pH value was gradually decreased to 6.0 retained for a long time (9 months) the capacity to resume growth on rich solid and liquid medium. Compared to vegetative cells, the ovoid forms exhibited an elevated resistance to heating (60–80°C) and antibiotics (hygromycin, kanamycin, and tetracycline). The ovoid forms of the M. smegmatis wild-type strain were classified as dormant forms based on their survival capacity, resistance to deleterious factors, and structural peculiarities. The ovoid forms generated in poststationary cultures upon decreasing the pH value to 5.0 or below lost the colony-forming capacity. It was established that the capacity to form ovoid cells upon gradual decrease in the pH value to 6.0 was reduced in ΔdevR and hlp-0 mutants compared to the wild-type strain (generation of 5–6 and 40% dormant forms, respectively) The amount of M. smegmatis dormant cells formed correlated with the acidification degree of the medium. The model developed can be used in tests of new antibacterial preparations that effectively inhibit resuscitating mycobacterial dormant forms that persist in the host organism.     

Exceptional carbon uptake in European forests during the warm spring of 2007: a data–model analysis

Temperate and boreal forests undergo drastic functional changes in the springtime, shifting within a few weeks from net carbon (C) sources to net C sinks. Most of these changes are mediated by temperature. The autumn 2006–winter 2007 record warm period was followed by an exceptionally warm spring in Europe, making spring 2007 a good candidate for advances in the onset of the photosynthetically active period. An analysis of a decade of eddy covariance data from six European forests stands, which encompass a wide range of functional types (broadleaf evergreen, broadleaf deciduous, needleleaf evergreen) and a wide latitudinal band (from 44° to 62°N), revealed exceptional fluxes during spring 2007. Gross primary productivity (GPP) of spring 2007 was the maximum recorded in the decade examined for all sites but a Mediterranean evergreen forest (with a +40 to +130 gC m^?2 anomaly compared with the decadal mean over the January–May period). Total ecosystem respiration (TER) was also promoted during spring 2007, though less anomalous than GPP (with a +17 to +93 gC m^?2 anomaly over 5 months), leading to higher net uptake than the long‐term mean at all sites (+12 to +79 gC m^?2 anomaly over 5 months). A correlative analysis relating springtime C fluxes to simple phenological indices suggested spring C uptake and temperatures to be related. The CASTANEA process‐based model was used to disentangle the seasonality of climatic drivers (incoming radiation, air and soil temperatures) and biological drivers (canopy dynamics, thermal acclimation of photosynthesis to low temperatures) on spring C fluxes along the latitudinal gradient. A sensitivity analysis of model simulations evidenced the roles of (i) an exceptional early budburst combined with elevated air temperature in deciduous sites, and (ii) an early relief of winter thermal acclimation in coniferous sites for the promotion of 2007 spring assimilation.     

Understanding Fluctuations in Bobcat Harvest at the Northern Limit of Their Range

ABSTRACT In Quebec, Canada, harvest of bobcats (Lynx rufus) started to decline in 1985 and by 1991, harvest seasons were closed due to concerns of a perceived population decline. Since the closing of harvest season in 1991, the average temperature has increased, snow quantity has decreased, and important changes in agriculture and forest management have occurred. In light of changing conditions, the situation of Quebec bobcats needed reassessment. Thus, we analyzed harvest data to clarify the current status of bobcat populations in Quebec. From 1980 to 1991, bobcat harvest in Quebec was strongly correlated with bobcat harvest in Maine (USA), Nova Scotia (Canada), Ontario (Canada), and Vermont (USA). Extrapolations of harvest in Quebec relative to harvest in Maine, Ontario, Vermont, and Nova Scotia suggested an increase in number of bobcats after 1991. Mass of male and female bobcats before 1991 was less than mass of animals captured after 1991. Percentage of juveniles in the reported harvest before 1991 was higher than after 1991. However, percentage of males and litter sizes in the harvest did not differ before and after 1991. The geographic distribution of bobcats captured has gradually expanded after the closure of the harvest season. Our findings suggest that bobcat populations in Quebec have recovered from the 1985–1991 decline, and that the harvest season for this species could resume. This study also illustrates how managers can rely on data from neighboring jurisdiction to manage species when local harvest data is unavailable.     

Antioxidant systems of moderately thermophilic methanotrophs <Emphasis Type="Italic">Methylocaldum szegediense</Emphasis> and <Emphasis Type="Italic">Methylococcus capsulatus</Emphasis>

Moderately thermophilic methanotrophs Methylocaldum szegediense O-12 and Methylococcus capsulatus Bath exhibit activities of antioxidant protection enzymes: glutathione peroxidase, superoxide dismutase, and cytochrome c peroxidase. The cells of methanotrophs grown at optimal temperatures (57 or 45°C, respectively) produce reactive oxygen species more actively than those grown at suboptimal temperatures, and exhibit higher activities of the membrane-associated cytochrome c peroxidase. Glutathione, glutathione peroxidase, and glucose-6-phosphate dehydrogenase levels in Md. szegediense O-12 increased in response to lowering of the cultivation temperature. By contrast, glutathione accumulation in cells of Mc. capsulatus Bath and the activity of glutathione peroxidase at a suboptimal temperature (29°C) were lower than at the optimal one. The role of the multilevel system of antioxidant protection in the adaptation of methanotrophs to temperature fluctuations is discussed.     

Utilization of milk energy by suckling mink kits

A total of 36 mink dams and their litters of 3, 6 or 9 kits were used for determination of milk intake of the suckling young by means of deuterium dilution technique, and chemical composition of milk and of kit bodies. Measurements were performed during lactation weeks 1?–?4, each week with 3 dams with each litter size. Milk intake was determined over a 48?h measurement period, and by the end of this milk samples were collected and 2 kits (litters of 6 and 9) or 1 kit per litter (litters of 3) were killed for body chemical composition. Based on the results, different models were applied for calculation of the energetic efficiency of milk. Dam milk yield increased steadily from week 1 until week 3 but only slightly from week 3 to 4. The increase declined with increasing litter size, and for dams suckling 9 kits the increment from week 3 to week 4 was only 2?g. The dry matter content of milk increased significantly as lactation progressed, being reflected in crude protein increasing from 6.9% in lactation week 1 to 8.1% in week 4. Milk fat increased concomitantly from 5.6% to 8.0%. In kit bodies, crude protein content increased from 9.4% in week 1 to about 12% in weeks 3 and 4. Body fat content increased from week 1 (4.1%) to week 3 (8.4%) and then declined in week 4 (7.1%). Animals suckled in litters of 3 kits had the highest milk intake and live weight and kits suckled in litters of 9 had the lowest milk intake, live weight and daily gain. In terms of milk intake per g gain kits in litters of 6 were the most efficient, with 4.1?g milk per g body gain. The metabolizable energy requirement for maintenance (MEm) was estimated to 448 kJ/kg^0.75 and the efficiency of utilization of ME for body gain (k_g) to 0.67, the estimates being higher (MEm) or in good agreement with previous findings (k_g) in suckling mink kits.     

Sulfate-reducing bacteria Desulfobulbus sp. strain BH from a freshwater lake in Guizhou Province, China

Sulfate-reducing bacteria (SRB) from the anaerobic sediments of the freshwater Baihua Lake (Guizhou Province, China) at eutrophicated sites polluted with heavy metals, particularly with mercury, have been studied. SRB belonging to the genus Desulfobulbus prevailed. A detailed study of the dominating Desulfobulbus isolate designated as strain BH revealed its difference in regards to genetic, morphological, physiological, and biochemical characteristics from the other species of this genus. Strain BH utilized a wide range of organic substrates, demonstrated a high level of hydrogen sulfide production, and could be considered an important component of the microbial community in the polluted lake.     

Effective PCR detection of vegetative and dormant bacterial cells due to a unified method for preparation of template DNA encased within cell envelopes

The unified method of template preparation for PCR in the form of DNA covered by permeabilized cell envelopes was used for the cells of different physiological status (vegetative, dormant forms of different types, and nonviable micromummies). The procedure for the preparation of template DNA included one-stage (boiling in a buffer with chaotropic salts) or two-stage (boiling in a buffer with chaotropic salts followed by treatment with proteinase K) sample preparation. The proposed method proved effective for detection of not only vegetative cells but also of the bacillary spores and the cystlike dormant cells (CLC) of non-spore-forming bacteria. For example, the two-stage sample preparation of Bacillus cereus spores resulted in the PCR sensitivity increase up to the detection level of 3–30 spores per sample; the one-stage sample preparation was three orders of magnitude less efficient (10₄ spores per sample). An increase in the sensitivity of PCR detection (4–10-fold) owing to the use of the two-stage sample preparation was shown for bacillary, staphylococcal, and mycobacterial CLC. The possibility of PCR detection of staphylococcal micromummies with irreversibly lost viability, which were therefore undetectable by plating techniques, was also demonstrated. The application of the unified sample preparation method ensuring efficacious PCR detection of bacterial cells, irrespective of their physiological state, may be a promising approach to more complete detection of microbial diversity and the overall insemination of natural substrates.