In vivo Model Experiment Using Laying Hens Treated with Enterococcus faecium EM41 from Ostrich Faeces and its Enterocin EM41

Enterococcus faecium EM41 is an isolate from ostrich faeces. It produces a thermo-stable proteinaceous substance, bacteriocin (enterocin) EM41 with the highest inhibition activity in late logarithmic phase of growth (25 600 AU/ml). This strain and its enterocin have not been previously tested in animals. Lohmann Brown laying hens (aged 45 weeks) were involved in this model/pilot experiment, divided into 3 groups 6 birds in each. E. faecium EM41 applied was a variant treated with rifampicin (109 cfu/ml, dose 400 μl/animal/day) to differentiate it from the other enterococci. Partially-purified enterocin EM41 (Ent EM41, dose 40 μl/animal/day) and its producer were applied to water for 21 days. The experiment lasted 35 days. Sampling was performed at days 0-1, 21 (3 weeks of additive application), 35 (2 weeks after cessation of additive application) from every bird. E. faecium EM41 sufficiently colonized the intestinal tract of laying hens from the initial concentration 109 cfu/g, its count reached 4.30 log cfu/g at day 21. PCR genotypization confirmed the identity of the EM41 strain with the species Enterococcus faecium. E. faecium EM41 and its enterocin EM41 showed antimicrobial effects demonstrated by reduction of coagulase-positive and coagulase-negative staphylococci, coliforms, Pseudomonas spp., Aeromonas spp. and Campylobacter spp. The hens were Salmonella spp. free. After administration of both additives, phagocytic activity was stimulated with a significant increase. The additives did not negatively influence biochemical and haematological parameters or weight gains.

Enterocin HZ produced by a wildEnterococcus faeciumstrain isolated from a traditional, starter-free pickled cheese

BacteriogenicEnterococcus faeciumHZ was identified by using biochemical (Strep-API 20, API-50 CHL, fatty acid profile) and 16S rRNA analysis (99·99 %).Ent. faeciumHZ was sensitive to clinically important antibiotics such as vancomycin, and did not have gelatinase and haemolysis activities. Enterocin HZ, a bacteriocin fromEnt. faeciumHZ, was sensitive to papain and tyripsin, but resistant to pepsin, lipase, catalase, α-amylase, organic solvents, detergents, ß-mercaptoethanol, and heat treatment (90 °C/30 min). It was biologically active at pH 2·0–9·0 and synthesised at the highest level in MRS or M17 broth at 32 or 37 °C with an inoculum amount of 0·1–0·5 % and an initial pH of 6·0–7·0. Enterocin HZ production reached maximum level at middle and late logarithmic phase and its molecular weight was ∼4·5 kDa. It was active against some Gram-positive foodborne bacteria.Ent. faeciumHZ or its bacteriocin enterocin HZ is a good candidate to be studied as a food biopreservative since enterocin HZ showed strong bactericidal activity againstListeria monocytogenesin UHT milk and alsoEnt. faeciumHZ grew very well in milk and produced enterocin HZ at maximum level.

MpeR Regulates themtrEfflux Locus in Neisseria gonorrhoeae and Modulates Antimicrobial Resistance by an Iron-Responsive Mechanism

ABSTRACTPrevious studies have shown that the MpeR transcriptional regulator produced byNeisseria gonorrhoeaerepresses the expression ofmtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation ofmpeRhas been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of themtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that themtrRgene, which encodes a direct repressor (MtrR) ofmtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of themtrCDEefflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression ofmtrRwas direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of themtrefflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.

Growth Phase- and Cell Division-Dependent Activation and Inactivation of the σ32 Regulon in Escherichia coli

ABSTRACT Alternative sigma factors allow bacteria to reprogram global transcription rapidly and to adapt to changes in the environment. Here we report on growth- and cell division-dependent σ32 regulon activity in Escherichia coli in batch culture. By analyzing σ32 expression in growing cells, an increase in σ32 protein levels is observed during the first round of cell division after exit from stationary phase. Increased σ32 protein levels result from transcriptional activation of the rpoH gene. After the first round of bulk cell division, rpoH transcript levels and σ32 protein levels decrease again. The late-logarithmic phase and the transition to stationary phase are accompanied by a second increase in σ32 levels and enhanced stability of σ32 protein but not by enhanced transcription of rpoH. Throughout growth, σ32 target genes show expression patterns consistent with oscillating σ32 protein levels. However, during the transition to early-stationary phase, despite high σ32 protein levels, the transcription of σ32 target genes is downregulated, suggesting functional inactivation of σ32. It is deduced from these data that there may be a link between σ32 regulon activity and cell division events. Further support for this hypothesis is provided by the observation that in cells in which FtsZ is depleted, σ32 regulon activation is suppressed.

Copper Extrusion after Accumulation during Growth of Copper-Tolerant Yeast Yarrowia lipolytica

The Cu2+-tolerant yeast Yarrowia lipolytica accumulated Cu2+ until the late logarithmic phase. Thereafter, Cu2+ was temperature-dependently extruded into phosphate-limited culture medium containing high concentrations of heavy metal ions but not into 10 mm 2-(Nmorpholino) ethane sulfonic acid (MES) buffer (pH 6.0). Peptone in the culture medium played an important role in the extrusion, which proceeded even when peptone was substituted with cysteine or histidine, but not with any other amino acid tested.

Expression of a Mitochondrial Peroxiredoxin Prevents Programmed Cell Death in Leishmania donovani

ABSTRACT Leishmania promastigote cells transmitted by the insect vector get phagocytosed by macrophages and convert into the amastigote form. During development and transformation, the parasites are exposed to various concentrations of reactive oxygen species, which can induce programmed cell death (PCD). We show that a mitochondrial peroxiredoxin (LdmPrx) protects Leishmania donovani from PCD. Whereas this peroxiredoxin is restricted to the kinetoplast area in promastigotes, it covers the entire mitochondrion in amastigotes, accompanied by dramatically increased expression. A similar change in the expression pattern was observed during the growth of Leishmania from the early to the late logarithmic phase. Recombinant LdmPrx shows typical peroxiredoxin-like enzyme activity. It is able to detoxify organic and inorganic peroxides and prevents DNA from hydroxyl radical-induced damage. Most notably, Leishmania parasites overexpressing this peroxiredoxin are protected from hydrogen peroxide-induced PCD. This protection is also seen in promastigotes grown to the late logarithmic phase, also characterized by high expression of this peroxiredoxin. Apparently, the physiological role of this peroxiredoxin is stabilization of the mitochondrial membrane potential and, as a consequence, inhibition of PCD through removal of peroxides.

Survival of Aspergillus fumigatus in Serum Involves Removal of Iron from Transferrin: the Role of Siderophores

ABSTRACT Aspergillus fumigatus is a filamentous fungus which can cause invasive disease in immunocompromised individuals. A. fumigatus can grow in medium containing up to 80% human serum, despite very low concentrations of free iron. The purpose of this study was to determine the mechanism by which A. fumigatus obtains iron from the serum iron-binding protein transferrin. In iron-depleted minimal essential medium (MEM), A. fumigatus growth was supported by the addition of holotransferrin (holoTf) or FeCl3 but not by the addition of apotransferrin (apoTf). Proteolytic degradation of transferrin by A. fumigatus occurred in MEM-serum; however, transferrin degradation did not occur until late logarithmic phase. Moreover, transferrin was not degraded by A. fumigatus incubated in MEM-holoTf. Urea polyacrylamide gel electrophoresis showed that in MEM-holoTf, holoTf was completely converted to apoTf by A. fumigatus. In human serum, all of the monoferric transferrin was converted to apoTf within 8 h. Siderophores were secreted by A. fumigatus after 8 h of growth in MEM-serum and 12 h in MEM-holoTf. The involvement of small molecules in iron acquisition was confirmed by the fact that transferrin was deferrated by A. fumigatus even when physically separated by a 12-kDa-cutoff membrane. Five siderophores were purified from A. fumigatus culture medium, and the two major siderophores were identified as triacetylfusarinine C and ferricrocin. Both triacetylfusarinine C and ferricrocin removed iron from holoTf with an affinity comparable to that of ferrichrome. These data indicate that A. fumigatus survival in human serum in vitro involves siderophore-mediated removal of iron from transferrin. Proteolytic degradation of transferrin may play a secondary role in iron acquisition.

Stimulatory effect of aerosil on algal growth

Unicellular green alga represents not only a convenient model for its biochemical and physiological studies but also a sensitive system to test the effects of various environmental factors. Algae cells of two strains, SA-3 strain (exsymbiotic from Paramecium bursaria) and Chlorella vulgaris c-27, were asynchronously cultured in the presence of 0.01% Aerosil A-300. Aerosil effects on algae were monitored at logarithmic and stationary phases of their growth by flow cytometry and microscopic counting of algal numbers. The growth patterns of algae were evaluated by their forward light scatter versus fluorescence of endogenous chlorophyll (FL3-height) signal distributions. Although aerosil itself did not cause any direct effects on algal morphology, it affected the growth patterns and the numbers of algae of both strains. Their growth patterns were remarkably altered in the late logarithmic phase cultures (6-day cultures). However, a significant increase of cell numbers was found in the stationary phase cultures (9- and 12-day cultures). While C. vulgaris c-27 demonstrated an increase of cell numbers by approximately 11% in the 9- and 12-day cultures, the amounts of SA-3 cells in the 9- and 12-days cultures were increased by 16% and 35%, respectively. Our study shows aerosil in its colloidal form stimulates proliferation of algae mainly via an acceleration of their life cycles. The stimulatory effect of silica on the growth of algae, the mechanism of which remains to be clarified, might have a practical (e.g., ecological) interest for regulation of algal expansion.Key words: aerosil, cloned algae, growth, forward light scatter, chlorophyll fluorescence.

Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0·65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28°C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0·6% agarose overlay containing G418 as the selective drug, on top of a 0·8% agar base. Transformation efficiencies were as high as 10−5 cells per μg of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of ‘shotgun transformation’ to generate libraries of T. cruzi recombinants.

The Cell Wall-Anchored Streptomyces reticuli Avicel-Binding Protein (AbpS) and Its Gene

ABSTRACT Streptomyces reticuli produces a 35-kDa cellulose-binding protein (AbpS) which interacts strongly with crystalline forms of cellulose (Avicel, bacterial microcrystalline cellulose, and tunicin cellulose); other polysaccharides are recognized on weakly (chitin and Valonia cellulose) or not at all (xylan, starch, and agar). The protein could be purified to homogeneity due to its affinity to Avicel. After we sequenced internal peptides, the corresponding gene was identified by reverse genetics. In vivo labelling experiments with fluorescein isothiocyanate (FITC), FITC-labelled secondary antibodies, or proteinase K treatment revealed that the anchored AbpS protrudes from the surfaces of the hyphae. When we investigated the hydrophobicity of the deduced AbpS, one putative transmembrane segment was predicted at the C terminus. By analysis of the secondary structure, a large centrally located α-helix which has weak homology to the tropomyosin protein family was found. Physiological studies showed that AbpS is synthesized during the late logarithmic phase, independently of the carbon source.