Phenotypic and genetic analysis of Enterobacter spp. from a Brazilian oligotrophic freshwater lake

We characterized a population of Enterobacter spp. of the Enterobacter cloacae complex isolated from an oligotrophic lake; most isolates were identified as E. cloacae. Fingerprinting polymerase chain reaction (PCR), along with morphological, biochemical, physiological, and plasmid profiles analyses, including antimicrobial susceptibility testing, were performed on 22 environmental isolates. Misidentification occurred when using the API 20E identification system. Analysis of 16S rDNA sequences confirmed the close relatedness between species of the E. cloacae complex. The tDNA PCR allowed the differentiation and identification of the E. cloacae isolates. Evaluation of genetic diversity by 16S rDNA sequence, tDNA, internal transcribed spacers, and enterobacterial repetitive intergenic concensus profiles revealed nearly identical isolates, although they exhibited different physiological and antimicrobial resistance profiles. Among the Enterobacter isolates, 96% were resistant to at least one antimicrobial; multiple resistance was also found at a high frequency (86%). The antimicrobials against which resistance was found most frequently were beta-lactams, chloramphenicol, and streptomycin. Plasmids were found in 21 of the 22 Enterobacter isolates. This confirms the conception that antibiotic resistance can occur in oligotrophic freshwater lake bacteria, which has important implications for public health.     

Seasonality of two gill monogeneans from two freshwater fish from an oligotrophic lake in northeast Finland

The seasonal occurrence of Dactylogyrus amphibothrium and Discocotyle sagittata from the gills of ruffe (Gymnocephalus cernuus) and whitefish (Coregonus acronius), respectively was studied in Lake Yli-Kitka, a large oligotrophic lake in Northeast Finland. The lake, located near the Arctic Circle, is ice-covered for 7-8 months of the year. The prevalence of D. amphibothrium infection was 70.7% and remained high throughout all size-classes of fish. The length distribution and developmental stages of the worms indicated two generations per year. The overwintering generation produces a summer generation which lives for only a few weeks and matures in July. Discocotyle sagittata has only one generation yearly. Its worms mature and produce eggs in July-August and recruitment of the new generation starts in the autumn. The prevalence of D. sagittata infection varied between 40% (in January) and 6.7% (in September); smaller fish were more heavily infected than larger fish.     

Potential rates of nitrification and denitrification in an oligotrophic freshwater sediment system

Potential rates of nitrification and denitrification were measured in an oligotrophic sediment system. Nitrification potential was estimated using the CO oxidation technique, and potential denitrification was measured by the acetylene blockage technique. The sediments demonstrated both nitrifying and denitrifying activity. E_h, O₂, and organic C profiles showed two distinct types of sediment. One type was low in organic C, had high O₂ and E_h, and had rates of denitrification 1,000 times lower than the other which had high organic C, low O₂, and low E_h. Potential nitrification and denitrification rates were negatively correlated with E_h. This suggests that environmental heterogeneity in denitrifier and nitrifier populations in oligotrophic sediment systems may be assessed using E_h before sampling protocols for nitrification or denitrification rates are established. There was no correlation between denitrification and nitrification rates or between either of these processes and NH₄ ⁺ or NO₃ ^– concentrations. The maximum rate of denitrification was 0.969 nmole N cm^–3 hour^–1, and the maximum rate of nitrification was 23.6 nmole cm^–3 hour^–1, suggesting nitrification does not limit denitrification in these oligotrophic sediments. Some sediment cores had mean concentrations of 6.0 mg O₂/liter and still showed both nitrification and denitrification activity.     

Microscale decoupling of sediment oxygen consumption and microbial biomass in an oligotrophic lake

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Anaerobic oxidation of methane in sediments of Lake Constance, an oligotrophic freshwater lake

Anaerobic oxidation of methane (AOM) with sulfate as terminal electron acceptor has been reported for various environments, including freshwater habitats, and also, nitrate and nitrite were recently shown to act as electron acceptors for methane oxidation in eutrophic freshwater habitats. Radiotracer experiments with sediment material of Lake Constance, an oligotrophic freshwater lake, were performed to follow 14CO2 formation from 14CH4 in sediment incubations in the presence of different electron acceptors, namely, nitrate, nitrite, sulfate, or oxygen. Whereas 14CO2 formation without and with sulfate addition was negligible, addition of nitrate increased 14CO2 formation significantly, suggesting that AOM could be coupled to denitrification. Nonetheless, denitrification-dependent AOM rates remained at least 1 order of magnitude lower than rates of aerobic methane oxidation. Using molecular techniques, putative denitrifying methanotrophs belonging to the NC10 phylum were detected on the basis of the pmoA and 16S rRNA gene sequences. These findings show that sulfate-dependent AOM was insignificant in Lake constant sediments. However, AOM can also be coupled to denitrification in this oligotrophic freshwater habitat, providing first indications that this might be a widespread process that plays an important role in mitigating methane emissions.     

General vs. local differentiation between two closely related white oak species

The geographical distribution of species differentiation throughout the natural range of two sympatric, closely related species of oaks, Quercus petraea (Matt) Liebl. and Quercus robur L., was investigated. By sampling species in pairs in different European regions, from Spain to Poland and Romania, the differentiation between the two species could be subdivided into general and local differentiation. Nine sequence characterized amplified region (SCAR) markers corresponding to genomic regions which discriminate the two species were analysed using single-strand conformation polymorphism (SSCP) or classical electrophoresis (double-stranded DNA on agarose gel) on PCR products providing, respectively, codominant and dominant markers. Similar levels of gene diversity ( H _E) within the two species were observed, varying generally from 0.3 to 0.5 for dominant markers and from 0.5 to 0.86 for codominant markers. SSCP loci exhibited numerous alleles that were differently involved in species differentiation. The geographical distribution of species differentiation is heterogeneous between the regions, the north-east populations exhibiting higher differentiation than the others. For most loci, general differentiation was higher than local differentiation and was interpreted as the result of historical causes, selection pressures, and intra- and interspecific gene flow.     

Genetic diversity of Microcystis cyanophages in two different freshwater environments

Bacteriophages rapidly diversify their genes through co-evolution with their hosts. We hypothesize that gene diversification of phages leads to locality in phages genome. To test this hypothesis, we investigated the genetic diversity and composition of Microcystis cyanophages using 104 sequences of Ma-LMM01-type cyanophages from two geographically distant sampling sites. The intergenetic region between the ribonucleotide reductase genes nrdA and nrdB was used as the genetic marker. This region contains the host-derived auxiliary metabolic genes nblA, an unknown function gene g04, and RNA ligase gene g03. The sequences obtained were conserved in the Ma-LMM01 gene order and contents. Although the genetic diversity of the sequences was high, it varied by gene. The genetic diversity of nblA was the lowest, suggesting that nblA is a highly significant gene that does not allow mutation. In contrast, g03 sequences had many point mutations. RNA ligase is involved in the counter-host’s phage defense mechanism, suggesting that phage defense also plays an important role for rapid gene diversification. The maximum parsimony network and phylogenic analysis showed the sequences from the two sampling sites were distinct. These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes.     

Biomass and annual production of the freshwater mussel Elliptio complanata in an oligotrophic softwater lake

SUMMARY. An extensive survey of Mirror Lake, New Hampshire, was carried out by divers with SCUBA to assess the importance of the freshwater mussel Elliptio complanata in this softwater lake ecosystem. Density (0.032 adults m⁻²), biomass (52 mg m⁻² as dry organic matter) and annual production (6.4 mg m⁻² as dry organic matter) of the mussel population are low when compared with results from other studies, corresponding with the general observation that mussels are scarce in soft, oligotrophic waters. We reject the traditional view that the low mussel density is a result of low calcium concentrations in Mirror Lake, and propose that mussel populations may be regulated by a scarcity of appropriate fish hosts in unproductive lakes. Elliptio complanata is probably not important in the metabolism or biochemistry of the Mirror Lake ecosystem.     

High genetic and physiological diversity of sulfate-reducing bacteria isolated from an oligotrophic lake sediment

The community structure of sulfate-reducing bacteria in littoral and profundal sediments of the oligotrophic Lake Stechlin (Germany) was investigated. A collection of 32 strains was isolated from the highest positive dilutions of most-probable-number series, and their partial 16S rRNA gene sequences and genomic fingerprints based on ERIC (enterobacterial repetitive intergenic consensus)-PCR were analyzed. The strains fell into eight distinct phylogenetic lineages, and the majority (70%) showed a close affiliation to the genus Desulfovibrio. Most of the remaining strains (22%) were related to the gram-positive Sporomusa and Desulfotomaculum groups. A high redundancy of 16S rRNA gene sequences was found within several of the phylogenetic lineages. This low phylogenetic diversity was most pronounced for the subset of strains isolated from oxic sediment layers. ERIC-PCR revealed that most of the strains with identical 16S rRNA gene sequences were genetically different. Since strains with identical 16S rRNA gene sequences but different genomic fingerprints also differed considerably with respect to their physiological capabilities, the high diversity detected in the present work is very likely of ecological relevance. Our results indicate that a high diversity of sulfate-reducing bacterial strains can be recovered from the natural environment using the established cultivation media. Received: 20 April 1998 / Accepted: 12 June 1998     

Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp.

An exopolysaccharide obtained from an alkaliphilic bacterium closely related to Bacillus spp. was found to contain D-galactopyranuronic acid (GalpA), 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose (QuipNAc4NAc), 2-acetamido-2-deoxy D-mannopyranuronic acid (ManpNAcA) and one uncommon unit of D-galactopyranuronic acid with the carboxyl group amide-linked to glycine [GalpA(Gly)]. The polysaccharide was studied by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy both on native polysaccharide and on monosaccharides and oligosaccharides obtained from methanolysis and from anhydrous HF solvolysis. The following linear structure of the repeating unit was established: -->3)-alpha-D-GalpA(Gly)-(1-->4)-beta-D-ManpNAcA-(1-->4)-alp ha-D-Galp A-(1-->3)-alpha-D-QuipNAc4NAc-(1-->. A preliminary phylogenetic assignment for the bacterium is also reported.     

Genome differentiation between two closely related species of Sideritis L. (Lamiaceae)

We have analysed the karyotype structure, DNA amounts and seed protein profiles of three isolated populations of two species: S. saetabensis and S. tragoriganum. These species are closely related from a karyological and morphological point of view. The differences in karyotypes and seed protein profiles between species and among populations could be the result of the differentiation that they are undergoing at present due to geographical isolation.     

Effects of temperature and sediment properties on benthic CO2 production in an oligotrophic boreal lake

1. Temperature and many other physical and chemical factors affecting CO₂ production in lake sediments vary significantly both seasonally and spatially. The effects of temperature and sediment properties on benthic CO₂ production were studied in in situ and in vitro experiments in the boreal oligotrophic Lake Pääjärvi, southern Finland. 2. In in situ experiments, temperature of the water overlying the shallow littoral sediment varied seasonally between 0.5 and 15.7 °C, but in deep water (≥20 m) the range was only 1.1–6.6 °C. The same exponential model (r² = 0.70) described the temperature dependence at 1.2, 10 and 20 m depths. At 2.5 and 5 m depths, however, the slopes of the two regression models (r² = 0.94) were identical but the intercept values were different. Sediment properties (wet, dry, mineral and organic mass) varied seasonally and with depth, but they did not explain a significantly larger proportion of variation in the CO₂ output rate than temperature. 3. In in vitro experiments, there was a clear and uniform exponential dependence of CO₂ production on temperature, with a 2.7‐fold increase per 10 °C temperature rise. The temperature response (slope of regression) was always the same, but the basic value of CO₂ production (intercept) varied, indicating that other factors also contributed to the benthic CO₂ output rate. 4. The annual CO₂ production of the sediment in Lake Pääjärvi averaged 62 g CO₂ m^?2, the shallow littoral at 0–3 m depth releasing 114 g CO₂ m^?2 and deep profundal (>15 m) 30 g CO₂ m^?2. On the whole lake basis, the shallow littoral at 0–3 m depth accounted for 53% and the sediment area in contact with the summer epilimnion (down to a depth c. 10 m) 75% of the estimated total annual CO₂ output of the lake sediment, respectively. Of the annual production, 83% was released during the spring and summer. 5. Using the temperature‐CO₂ production equations and climate change scenarios we estimated that climatic warming might increase littoral benthic CO₂ production in summer by nearly 30% from the period 1961–90 to the period 2071–2100.     

Population dynamics of bacterioplankton in an oligotrophic lake

The population ecology of bacterioplankton was studied overa 3 year period in Mirror Lake, an oligotrophic lake in thenortheastern USA. Bacterial population density, biomass, andrates of biomass production in the epilimnion and hypolimnionwere examined for their relationship with several environmentalparameters. Bacterioplankton density fluctuated between 0.5and 7 x l0 bacteria ml^–1, with highest values in the anoxichypolimnion. At all depths there was a trend towards a higherdensity of bacteria from spring to midsummer, followed by adecline in late summer to early autumn. Cocci tended to dominatebacterial cell shapes from winter to midsummer, after whichrod-shaped cells became most abundant. Rod-shaped cells contributedthe most to bacterioplankton biomass at all depths and timesof year. The mean annual biovolume of all bacterioplankton was0.12 µ cell^–1. The mean annual areal bacterioplanktonbiomass was 11–12 mmol C m^–2. The percentage ofbacterial to phytoplankton biomass per volume in summertimewas 27% in the epilimnion and 11% in the hypolimnion. Averageannual and summertime bacterial production estimated using the[3H]thymidine method was similar to previous estimates of bacterialproduction measured in Mirror Lake using other methods. Theaverage ratio of bacterial to net phytoplankton production pervolume was 0.34 in the epilimnion, and between 0.65 and 1 1.depending on depth, in the hypolimnion during summer. Of severalvariables considered in regression analyses, only temperatureexplained >50% of the variance in bacterial production inboth the hypolimnion and epilimnion. Above 14°C, however,bacterial production and growth rate in the epilimnion werenot clearly related to temperature. During the period of midsummerhypolimnetic anoxia, despite colder temperatures in the hypolimnion,bacterial production was up to 10 times greater than in theepilimnion.     

The characterization of amidohydrolases in a freshwater lake sediment

The properties of three amidohydrolases, i.e., urease (I) EC 3.5.1.5, L-asparaginase (II) EC 3.5.1.1, and L-glutaminase (III) EC 3.5.1.2, were studied in sediment samples taken from a shallow eutrophic freshwater lake.Sediment samples were air dried (ADS) and stored for at least 3 months before being enzymically characterized. The pH optimum of I, II, and III were pH 7.0, 8.4, and 6.5–7.0, respectively, while III in soluble extracts from ADS was most active between pH 8.0 and 9.0. The temperature response of the three enzymes in ADS gave E_a values of 38.9, 41.6, and 35.9 kJmol^–1 for I, II, and III, respectively. K_m and V_max values for ADS I, II, and III were 1.2 mM and 1.9mol NH₃ g^–1h^–1; 0.8 mM and 4.1mol NH₃ g^–1h^–1; and 1.25 mM and 17.4mol NH₃ g^–1h^–1. K_m values for all three enzymes in ADS extracts were at least an order of magnitude greater than those of the ADS. The susceptability of each enzyme to proteolysis was followed in ADS and fresh wet sediment and compared with that of III in an ADS extract. All sediment enzymes were found to be more resistant than the commercial preparation of bacterial L-glutaminase subjected to the same treatment. These results suggested that I, II, and III all exist to some extent as colloid-immobilized enzyme fractions in freshwater sediments and are analogous to the stable enzyme fractions in soils.     

Estimation of methanotroph abundance in a freshwater lake sediment

The numbers of methane-oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture-independent methods. Quantitative slot-blot hybridizations were performed with type I and type II methanotroph-specific probes. These data were compared to data from quantitative hybridizations using a pmoA-specific probe and a eubacterial probe. From the combined hybridization data, the methanotroph population in Lake Washington was estimated to be 3.6 x 10(8)-7.4 x 10(8) cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1 omega 8) and type II (18:1 omega 8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 x 10(8)-9.4 x 10(9) cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 x 10(8)-1.2 x 10(9) cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two- to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.     

Factors affecting the contribution by epiphytic algae to the primary productivity of an oligotrophic freshwater lake

A diatom-dominated population of epiphytic algae was studied in an oligotrophic lake to determine the factors which limit epiphyte growth and to measure their contribution to primary productivity. Algae were collected from plants growing at four sites in Lake George, N.Y., during the spring, summer, and fall of 1974. Samples were taken from 3 m, corresponding to the depth at which macrophytes were most productive. Algae exhibited an optimum temperature for H¹⁴CO₃^- uptake at 30 C, although the summer littoral lake temperature ranged from 18 to 25 C. Light saturation occurred at an intensity of 8,608 lux, approximating the environmental intensity at the depth from which algae were taken. Epiphytes exhibited their maximum photosynthetic capacity of 0.6 mg of carbon fixed/m² of macrophyte surface area per h in the early afternoon in mid-August. They assimilated approximately 5% as much inorganic carbon as the macrophytes from which they were taken. Epiphyte population densities followed the seasonal growth patterns of the macrophytes, with maximal leaf colonization remaining essentially constant relative to the leaf position on the plant. There was little change in density between sampling sites at any given time. Productivities of epiphytes from bottom leaves were 10-fold greater than those of epiphytes from top leaves. Addition of PO₄^-3, NO₃^-, NH₃, Si, and SO₄^-2 had no stimulatory effect on photosynthesis. Addition of HCO₃^- stimulated photosynthesis greater than 30%, suggesting that carbon may be a limiting nutrient for epiphytic algae in Lake George.     

Physical control of Eurasian watermilfoil in an oligotrophic lake

The introduction of Eurasian watermilfoil (Myriophyllum spicatum) into oligotrophic waters of high water clarity in temperate zones of North America has produced growth in excess of 6 m depth and yearly biomass approaching 1000 g m^–2 dry weight. From its initial observation in Lake George, New York, USA in 1985, by 1993 milfoil had spread to 106 discrete locations within the lake. A 7-year study of one site having no management showed milfoil to grow expansively, suppressing native plant species from 20 in 1987 to 6 in 1993 with the average number of species m^–2 quadrat declining from 5.5 in 1987 to less than 2 in 1993. Management of milfoil by means of hand harvesting, suction harvesting and benthic barrier has reduced the number of unmanaged sites from 106 in 1993 to 11. One year post-treatment at sites utilizing suction harvesting, showed a greater number of native species at all sites than pretreatment with a substantial reduction in milfoil biomass. At sites where benthic barrier was removed 1–2 years after installation, milfoil had recolonized 44% of grid squares within 30 days. Ninety days after barrier removal 74% of grid squares contained milfoil and one year later 71% of the grids supported milfoil. During the first year following mat removal, the average number of species m^–2 peaked at 4.7 and stabilized at 4.5 during the second year. Hand harvesting by SCUBA in areas of limited milfoil growth (new sites of infestation and sites of former treatment) was found to reduce the number of milfoil plants present in subsequent years. Hand harvesting did not eliminate milfoil at any of the sites and regrowth/colonization necessitated reharvesting every 3 or more years. Results of evaluations of physical plant management techniques indicate that (1) an integrated program utilizing different techniques based on plant density reduced the growth of milfoil and (2) long term commitment to aquatic plant management is necessary since none of the techniques employed singly were found to eliminate milfoil.     

Nitrogen-fixation in the littoral benthos of an oligotrophic lake

Blue-green algae are common in the benthos of Mirror Lake, New Hampshire (U. S. A.) — on macrophytes and on the lake bottom-and are probably responsible for the variable, sometimes high rates of N-fixation that detected by a series of acetylene-reduction assays during September and October.     

Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.     

The evolution of different maternal investment strategies in two closely related desert vertebrates

We compared egg size phenotypes and tested several predictions from the optimal egg size (OES) and bet‐hedging theories in two North American desert‐dwelling sister tortoise taxa, Gopherus agassizii and G. morafkai, that inhabit different climate spaces: relatively unpredictable and more predictable climate spaces, respectively. Observed patterns in both species differed from the predictions of OES in several ways. Mean egg size increased with maternal body size in both species. Mean egg size was inversely related to clutch order in G. agassizii, a strategy more consistent with the within‐generation hypothesis arising out of bet‐hedging theory or a constraint in egg investment due to resource availability, and contrary to theories of density dependence, which posit that increasing hatchling competition from later season clutches should drive selection for larger eggs. We provide empirical evidence that one species, G. agassizii, employs a bet‐hedging strategy that is a combination of two different bet‐hedging hypotheses. Additionally, we found some evidence for G. morafkai employing a conservative bet‐hedging strategy. (e.g., lack of intra‐ and interclutch variation in egg size relative to body size). Our novel adaptive hypothesis suggests the possibility that natural selection favors smaller offspring in late‐season clutches because they experience a more benign environment or less energetically challenging environmental conditions (i.e., winter) than early clutch progeny, that emerge under harsher and more energetically challenging environmental conditions (i.e., summer). We also discuss alternative hypotheses of sexually antagonistic selection, which arise from the trade‐offs of son versus daughter production that might have different optima depending on clutch order and variation in temperature‐dependent sex determination (TSD) among clutches. Resolution of these hypotheses will require long‐term data on fitness of sons versus daughters as a function of incubation environment, data as yet unavailable for any species with TSD.