Bacteria and viruses in the water column of tropical freshwater reservoirs

In tropical freshwater reservoirs of Sri Lanka, which are linked in an aquatic network, bacterial abundance and production as well as virus abundance, frequency of viral infection and virus production were investigated together with a set of nutrient species (Kjeldahl-N, NO3-N, total P, soluble P, PO4-P). At two characteristic seasons (wet season, dry season), samples were taken from two types of reservoirs (new upland impoundment and ancient, shallow lowland reservoir), each during 4 days at various depths of the entire water columns. Kjeldahl-N and total P were greatly elevated in the wind-mixed water body of the shallow impoundment during the dry season, whereas the deeper reservoir type exhibited no obvious seasonality. In SYBR green trade mark -stained samples, bacterial abundance showed no seasonal pattern in either reservoir type. Bacterial secondary production, however, was significantly elevated in the entire water column of the shallow impoundment under wind-mixed conditions in the dry season. Highest abundance of virus particles and elevated frequency of bacteria containing mature phages were also observed in the shallow reservoir during the dry season indicating favourable conditions for virus propagation. Data from this aquatic network show that most virus parameters, such as abundance or frequency of visibly infected cells, were positively linked to bacterial abundance and production, but also to organic nitrogen or some phosphorus species. We calculated that between 13.2% and 46.1% of the bacterial standing stocks would be subjected to virus-mediated mortality. Estimates of bacteriophage production revealed that from 10 x 10(9) up to 98 x 10(9) phages were produced per litre and day. Bacteria and viruses in the studied tropical freshwater system appear to be linked to various environmental conditions and may affect processes at the ecosystem scale.     

Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli

The mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large-scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02-1.2 microM. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB-ExbB-ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonB- and energy-dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by iron-siderophore receptors, including FepA, which promote its import across the outer membrane via a TonB- and energy-dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492.     

Structural and functional characterisation of the Toll like receptor 9 of Aotus nancymaae, a non-human primate model for malaria vaccine development

In the absence of suitable rodent animal models for Plasmodium falciparum malaria, the efficacy testing of asexual blood-stage vaccine candidates in Aotus nancymaae represents a tool to select between different formulations before conducting expensive human clinical trials. CpG oligonucleotides (ODN) specifically promote the production of pro-inflammatory and Th1-type cytokines and they enhance the immunogenicity of co-administered antigens. Toll like receptor 9 (TLR-9) binds directly and sequence-specifically to single-stranded un-methylated CpG-DNA mediating the biological effects of CpG ODN. We cloned and functionally characterised the TLR-9 cDNA of A. nancymaae. The cDNA encompassed 3,099 bp predicted to code for 1,032 amino acid residues. Results of homology searches to human TLR-9 suggested that the receptor is 93 and 94% identical at the nucleotide and amino acid sequence levels, respectively. Stimulation of splenocytes of A. nancymaae with CpG ODN resulted in proliferative responses in all animals analysed. FACS analysis of cultures incubated with CpG ODN 2006 indicated that the B cell marker CD20 was up-regulated consistent with B cell activation. The high level of sequence conservation of Aona-TLR-9 reinforces the suitability of A. nancymaae as animal model for malaria subunit vaccine development.The nucleotide sequence has been submitted to the GenBank nucleotide sequence database under the accession number AY788894.     

Evaluation of recA sequencing for the classification of Aeromonas strains at the genotype level

Aims: To evaluate the usefulness of partial recA sequences for the identification of Aeromonas strains at the genotype level. Methods and Results: A partial recA sequence was obtained from 21 type or reference strains and 33 Aeromonas isolates, collected in the South of Switzerland from human, animal and aquatic environments. The 272 bp long recA fragments showed a mean interspecies divergence of 7·8% and allowed the classification of strains at genotype level. However, some discrepancies could be observed with other gene sequence based analyses in the classification of some strains. Conclusions: The 272 bp long recA fragment is a good molecular marker to infer taxonomy of members of the genus Aeromonas, even if the primers we chose for the amplification did not allow its direct sequencing. Significance and Impact of the Study: In the genus Aeromonas, nucleotide sequences of some protein‐encoding genes have already been evaluated as molecular markers to be used in taxonomical and epidemiological researches. This study suggests the usefulness of a recA fragment as a further sequence to investigate for these purposes.     

Immunochemical investigation of four Saprolegnia species with parasitic activity in fish: Serological and kinetic characterization of a chymotrypsin-like activity

The antigenic structure of four aquatic Phycomycetes of the genusSaprolegnia with parasitic activity in fish was analyzed with immunoelectrophoresis and double diffusion techniques. It is shown that these serological tests might represent a valuable criterion for the identification of the fourSaprolegnia species:S. parasitica, S. ferax, S. delica, andS. diclina. A specific chromogenic reaction revealed the presence of a protease with chymotrypsin-like activity among the precipitation arcs obtained after immunoelectrophoresis. Analysis of this proteolytic activity by kinetic assay methods confirmed the results of the serological tests. Moreover estimates of the quantity of enzyme present in the cellular extract and in the culture medium were obtained. A possible relationship between the proteolytic activity and the host-parasite interaction was discussed.     

Predictors of Operative Mortality for Coronary Bypass Grafting in Patients with Ischemic Heart Disease

Predictors for operative mortality (OM) were studied in 172 consecutive patients (pts) undergoing coronary artery grafts (CAG) for angina pectoris.Seventy eight pts had Class IV angina; of the 147 patients given propranolol, 41 were gradually withdrawn from propranolol and finally discontinued 24 hours before surgery, and 106 were abruptly withdrawn from propranolol 24 hours before CAG; 20 pts had left main coronary disease; 156 pts had cardiopulmonary bypass (CPB) time shorter than 20 minutes, and 16 pts had a CPB longer than 120 minutes.The operative mortality was 5.2% (9/172) for the entire group. Class IV angina (OM 7%), abrupt propranolol withdrawal (OM 6.6%), left main coronary artery disease (OM 25%), and CPB longer than 120 minutes (OM 50%), all significantly increased OM. These variables were interdependent, however, as many pts belonged to several predictor categories, combinations of predictors were examined, in order to more accurately predict the risk of individual pts. The combination of left main coronary artery disease and CPB longer than 120 minutes; and Class IV angina and CPB longer than 120 minutes were significantly associated with higher operative mortality.We conclude that Class IV angina, abrupt propranolol withdrawal, left main coronary artery disease and prolonged CPB are potent, interdependent predictors of OM in pts undergoing CAG. Consideration of these predictors, alone and in combination, allows effective prediction of OM for CAG in patients with stable angina pectoris.     

Signature region within the 16S rDNA sequences of Aeromonas popoffii

To identify a group of eight Aeromonas strains of our collection showing ribotyping patterns similar to those described for the species Aeromonas popoffii, 16S rRNA gene sequence analysis was performed. Results were in agreement with the DNA binding values, and allowed the identification of a 'signature region' differentiating the A. popoffii strains from all other members of the genus Aeromonas.     

Dominance of a clonal green sulfur bacterial population in a stratified lake

For many years, the chemocline of the meromictic Lake Cadagno, Switzerland, was dominated by purple sulfur bacteria. However, following a major community shift in recent years, green sulfur bacteria (GSB) have come to dominate. We investigated this community by performing microbial diversity surveys using FISH cell counting and population multilocus sequence typing [clone library sequence analysis of the small subunit (SSU) rRNA locus and two loci involved in photosynthesis in GSB: fmoA and csmCA ]. All bacterial populations clearly stratified according to water column chemistry. The GSB population peaked in the chemocline ( c . 8 × 10⁶ GSB cells mL⁻¹) and constituted about 50% of all cells in the anoxic zones of the water column. At least 99.5% of these GSB cells had SSU rRNA, fmoA , and csmCA sequences essentially identical to that of the previously isolated and genome-sequenced GSB Chlorobium clathratiforme strain BU-1 (DSM 5477). This ribotype was not detected in Lake Cadagno before the bloom of GSB. These observations suggest that the C. clathratiforme population that has stabilized in Lake Cadagno is clonal. We speculate that such a clonal bloom could be caused by environmental disturbance, mutational adaptation, or invasion.     

Virus ecology of fluvial systems: a blank spot on the map?

The ecology of viruses has been studied only in a limited number of rivers and streams. In light of a recent re‐appraisal of the global fluvial surface area, issues such as abundance and production, host mortality and the influence of suspended particles and biofilms are addressed. Viral life cycles, potential impacts of viruses on water biochemistry and carbon flow, and viral diversity are considered. Variability in trophic levels along with the heterogeneous nature and hydrological dynamics of fluvial environments suggest a prevailingly physical control of virus‐related processes under lotic conditions and more biological control under lentic conditions. Viral lysis likely contributes to a pool of rapidly cycling carbon in environments typically characterized by high proportions of recalcitrant terrestrial carbon. On average, 33.6% (equalling 0.605 Pg C year^?1) of the globally respired carbon from fluvial systems may pass through a viral loop. Virus distribution and the proportion of organic material in horizontal transport versus processes in retention zones remain to be determined in detail. The need for up‐scaling the contribution of virus‐related processes in fluvial systems is of global relevance. Further, the role of climate change and the effect of anthropogenic alterations of fluvial systems on viruses require attention. The identification of these considerable knowledge gaps should foster future research efforts.