Mercury decreases culturability of Pseudomonas frederiksbergensis JAJ 28 in soil microcosms

Mercury is a biologically potent heavy metal, which has been found to change the diversity of culturable bacteria. Therefore, we investigated whether Hg kills bacteria in soil or reduces culturability. Soil microcosms were inoculated with Pseudomonas frederiksbergensis JAJ 28 and were sampled regularly during 28 days. The total number of acridine orange-stained cells was relatively constant, and Hg reduced the number on only one sampling day. However, the fraction of culturable cells on 1/10 tryptic soy agar was lowered on days 6, 13, and 21. The number of microcolony forming units, which represents viable cells, was also affected by Hg, but this effect was delayed compared with the effects on CFUs. The amount of headspace CO2 per cell was overall increased by Hg, another indication of the toxic effects of Hg on the bacterial cells. Our results thus emphasize the need to take culturability into account when studying the effects of heavy metals on bacterial diversity.     

Visual clone identification of Penicillium commune isolates

A method for visual clone identification of Penicillium commune isolates was developed. The method is based on images of fungal colonies acquired after growth on a standard medium and involves a high degree of objectivity, which in future studies will make it possible for non-experts to perform a qualified identification of different species as well as clones within a species. A total of 77 P. commune isolates from a cheese dairy were 3-point inoculated on Yeast Extract Sucrose (YES) agar and incubated for 7 days at 25 degrees C. After incubation, the isolates were classified into groups containing the same genotype determined by DNA fingerprinting (AFLP). Each genotype also has a specific phenotype such as different colony colours. By careful image acquisition, colours were measured in a reproducible way. Prior to image analysis, each image was corrected with respect to colour, geometry and self-illumination, thereby gaining a set of directly comparable images. A method for automatic extraction of a given number of concentric regions was used. Using the positions of the regions, a number of relevant features--capturing colour and colour-texture from the surface of the fungal colonies--was extracted for further analysis. We introduced the Jeffreys-Matusitas (JM) distance between the feature distributions to express the similarity between regions in two colonies, and to evaluate the overall (weighted) similarity. The nearest neighbour (NN) classification rule was used. On a dataset from 137 isolates, we obtained a "leave-one-out" cross-validation identification rate of approximately 93-98% compared with the result of DNA fingerprinting.     

Interaction between alginates and manganese cations: identification of preferred cation binding sites

Algal and bacterial alginates have been studied by means of 13C NMR spectroscopy in presence of paramagnetic manganese ions in order to reveal the nature of their interaction with bivalent cations. It is found that the mannuronate blocks bind manganese cations externally near their carboxylate groups, while guluronate blocks show the capability to integrate Mn2+ into pocket-like structures formed by adjacent guluronate residues. In alternating mannuronate-guluronate blocks, manganese ions preferentially locate in a concave structure formed by guluronate-mannuronate pairs. Partial acetylation of the alginate generally reduces its capability to interact with bivalent cations, however, the selectivity of the binding geometry is conserved. The results may serve as a hint for the better understanding of the alginate gelation in presence of calcium ions.     

Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.     

Involvement of tyrosines 114 and 139 of subunit NuoB in the proton pathway around cluster N2 in Escherichia coli NADH:ubiquinone oxidoreductase

The proton-pumping NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron transfer is accomplished by FMN and a series of iron-sulfur clusters. Its coupling with proton translocation is not yet understood. Here, we report that the redox reaction of the FeS cluster N2 located on subunit NuoB of the Escherichia coli complex I induces a protonation/deprotonation of tyrosine side chains. Electrochemically induced FT-IR difference spectra revealed characteristic tyrosine signals at 1,515 and 1,498 cm(-1) for the protonated and deprotonated form, respectively. Mutants of three conserved tyrosines on NuoB were generated by complementing a chromosomal in-frame deletion strain with nuoB on a plasmid. Though the single mutations did not alter the electron transport activity of complex I, the EPR signal of cluster N2 was slightly shifted. The tyrosine signals detected by FT-IR spectroscopy were roughly halved in the mutants Y114C and Y139C while only minor changes were detected in the Y154H mutant. The enzymatic activity of the Y114C/Y139F double mutant was 80% reduced, and FT-IR difference spectra of the double mutant revealed a complete loss the modes characteristic for protonation reactions of tyrosines. Therefore, we propose that tyrosines 114 and 139 on NuoB were protonated upon reduction of cluster N2 and were thus involved in the proton-transfer reaction coupled with its redox reaction.     

Population dynamics of active and total ciliate populations in arable soil amended with wheat

Soil protozoa are characterized by their ability to produce cysts, which allows them to survive unfavorable conditions (e.g., desiccation) for extended periods. Under favorable conditions, they may rapidly excyst and begin feeding, but even under optimal conditions, a large proportion of the population may be encysted. The factors governing the dynamics of active and encysted cells in the soil are not well understood. Our objective was to determine the dynamics of active and encysted populations of ciliates during the decomposition of freshly added organic material. We monitored, in soil microcosms, the active and total populations of ciliates, their potential prey (bacteria and small protozoa), their potential competitors (amoebae, flagellates, and nematodes), and their potential predators (nematodes). We sampled with short time intervals (2 to 6 days) and generated a data set, suitable for mathematical modeling. Following the addition of fresh organic material, bacterial numbers increased more than 1,400-fold. There was a temporary increase in the number of active ciliates, followed by a rapid decline, although the size of the bacterial prey populations remained high. During this initial burst of ciliate growth, the population of cystic ciliates increased 100-fold. We suggest that internal population regulation is the major factor governing ciliate encystment and that the rate of encystment depends on ciliate density. This model provides a quantitative explanation of ciliatostasis and can explain why protozoan growth in soil is less than that in aquatic systems. Internally governed encystment may be an essential adaptation to an unpredictable environment in which individual protozoa cannot predict when the soil will dry out and will survive desiccation only if they have encysted in time.     

Increased or decreased levels of Caenorhabditis elegans lon-3, a gene encoding a collagen,cause reciprocal changes in body length

Body length in C. elegans is regulated by a member of the TGFbeta family, DBL-1. Loss-of-function mutations in dbl-1, or in genes encoding components of the signaling pathway it activates, cause worms to be shorter than wild type and slightly thinner (Sma). Overexpression of dbl-1 confers the Lon phenotype characterized by an increase in body length. We show here that loss-of-function mutations in dbl-1 and lon-1, respectively, cause a decrease or increase in the ploidy of nuclei in the hypodermal syncytial cell, hyp7. To learn more about the regulation of body length in C. elegans we carried out a genetic screen for new mutations causing a Lon phenotype. We report here the cloning and characterization of lon-3. lon-3 is shown to encode a putative cuticle collagen that is expressed in hypodermal cells. We show that, whereas putative null mutations in lon-3 (or reduction of lon-3 activity by RNAi) causes a Lon phenotype, increasing lon-3 gene copy number causes a marked reduction in body length. Morphometric analyses indicate that the lon-3 loss-of-function phenotype resembles that caused by overexpression of dbl-1. Furthermore, phenotypes caused by defects in dbl-1 or lon-3 expression are in both cases suppressed by a null mutation in sqt-1, a second cuticle collagen gene. However, whereas loss of dbl-1 activity causes a reduction in hypodermal endoreduplication, the reduction in body length associated with overexpression of lon-3 occurs in the absence of defects in hypodermal ploidy.     

Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate (³⁵SO₄²⁻). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a ³⁵S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO₄²⁻) of 10⁻¹⁴ to 10⁻¹³ mol of SO₄²⁻ cell⁻¹ day⁻¹ were calculated, which is within the range of qSO₄²⁻ values previously reported for pure cultures of SRB (10⁻¹⁵ to 10⁻¹⁴ mol of SO₄²⁻ cell⁻¹ day⁻¹). qSO₄²⁻ values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 × 10⁻¹⁰ to 7 × 10⁻¹⁰ mol of SO₄²⁻ cell⁻¹ day⁻¹), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.