Growth of Acanthamoeba castellanii and Hartmannella vermiformis on live, heat-killed and DTAF-stained bacterial prey

The growth responses of two species of amoeba were evaluated in the presence of live, heat-killed and heat-killed/5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)-stained cells of Escherichia coli, Pseudomonas aeruginosa, Klebsiella aerogenes, Klebsiella ozaenae and Staphylococcus aureus. The specific growth rates of both species were significantly higher with live bacterial prey, the only exception being Hartmannella vermiformis feeding on S. aureus, for which growth rates were equivalent on all prey states. There was no significant difference between growth rates, yield or ingestion rates of amoebae feeding on heat-killed or heat-killed/stained bacterial cells, suggesting that it was the heat-killing process that influenced the amoeba-bacteria interaction. Pretreatment of prey cells had a greater influence on amoebic processing of Gram-negative bacteria compared with the Gram-positive bacterium, which appeared to be as a result of the former cells being more difficult to digest and/or losing their ability to deter amoebic ingestion. These antipredatory mechanisms included microcolony formation in P. aeruginosa, toxin production in K. ozaenae, and the presence of an intact capsule in K. aerogenes. E. coli and S. aureus did not appear to possess an antipredator mechanism, although intact cells of the S. aureus were observed in faecal pellets, suggesting that any antipredatory mechanism was occurring at the digestion stage.     

Effects of bacterial prey species and their concentration on growth of the amoebae Acanthamoeba castellanii and Hartmannella vermiformis

Two amoebae were presented with six bacterial prey at a range of concentrations, and the growth parameters of the amoebae were deduced. All but one bacterium (Synechococcus) resulted in a positive growth response, but the gram-positive bacterium Staphylococcus aureus proved to be difficult to digest and the heavily pigmented bacterium Klebsiella ozaenae induced unusual amoebic behavior prior to ingestion.     

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.     

Use of glucose starvation to limit growth and induce protein production in Escherichia coli

The use of glucose starvation to uncouple the production of recombinant beta-galactosidase from cell growth in Escherichia coli was investigated. A lacZ operon fusion to the carbon starvation-inducible cst-1 locus was used to control beta-galactosidase synthesis. beta-Galactosidase induction was observed only under aerobic starvation conditions, and its expression continued for 6 h following the onset of glucose starvation. The cessation of beta-galactosidase expression closely correlated with the exhaustion of acetate, an overflow metabolite of glucose, from the culture medium. Our results suggest the primary role of acetate in cst-1-controlled protein expression is that of an energy source. Using this information, we metered acetate to a glucose-starved culture and produced a metabolically sluggish state, where growth was limited to a low linear rate and production of recombiant beta-galactosidase occurred continuously throughout the experiment. The cst-1 controlled beta-galactosidase synthesis was also induced at low dilution rates in a glucose-limited chemostat, suggesting possible applications to high-density cell systems such as glucose-limited recycle reactors. This work demonstrates that by using an appropriate promoter system and nutrient limitation, growth can be restrained while recombinant protein production is induced and maintained.     

Effect of oxygen limitation and starvation on the benzalkonium chloride susceptibility of Escherichia coli

Aims: To investigate the effect of oxygen limitation, glucose-starvation and temperature on the susceptibility of Escherichia coli towards the quaternary ammonium biocide benzalkonium chloride (BAC). Methods and Results: The effect of BAC on planktonic and sessile cells were investigated using the gfp-tagged E. coli K-12 strain MG1655[pOX38Km]. Increasing temperature from 10°C to 30°C increased the bactericidal effect of BAC for both starved and nonstarved E. coli under aerobic and anaerobic conditions. The lowest minimum bactericidal concentration was observed for cells in anaerobic media at 30°C (30 mg l⁻¹ BAC). Decreasing cell densities increased the decay rate for BAC-exposed cells for both starved and nonstarved E. coli. Biofilms of E. coli exposed to BAC in anaerobic medium showed a greater percentage of membrane-compromised cells than biofilms grown in aerobic medium. Image analyses of BAC-exposed biofilms showed that membrane-compromised cells were occasionally located in the interior structure of the biofilm microcolonies. Conclusions: Increasing temperatures and the absence of oxygen, and energy substrates increased the antimicrobial effect of BAC towards E. coli. Significance and Impact of the Study: The results are relevant for understanding the disinfection efficacy of quaternary ammonium compounds towards planktonic and sessile bacteria.     

Noncoordinate control of the synthesis of different species of RNA in Escherichia coli K12 during uridine starvation

During uridine starvation inEscherichia coli K12, the rate of RNA accumulation comes down to about 7% of the nonstarved rate. This is achieved, in part, by an eight-fold increase in the assembly time of stable RNA molecules. However, the assembly time of mRNA molecules is not enhanced as much, being longer by a factor of 3 in starved cells compared to nonstarved ones. It, therefore, appears that the rate of synthesis of these two RNA species is noncoordinately controlled during uridine starvation. This control does not seem to be mediated by guanosine tetraphosphate.     

Morphology, growth and reversion in a stable L-form of Escherichia coli K12

An L-form isolated from Escherichia coli K12 by sequential treatment with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme was adapted to grow in hyperosmolar liquid cultures. It was stable in the absence of antibiotic when cultured in brain heart infusion (BHI) broth containing NaCl and CaCl2, the optimal concentrations being 0.34 M and 1 mM, respectively. No growth of the L-form was observed when CaCl2 was not added to BHI medium containing 0.34 M-NaCl. On the other hand, when KCl replaced NaCl as the osmotic stabilizer, growth of the L-form was repressed in the presence of CaCl2. Electron microscopy of the L-form confirmed the absence of a cell wall. A revertant strain derived from the L-form grew as a stable bacillary form in BHI medium without osmotic stabilizer. The growth characteristics of the revertant strain resembled those of the parent strain. The revertant strain produced L-forms in the presence of NaCl.     

Iron uptake and iron limited growth of Escherichia coli K-12

Cells of Escherichia coli K-12 could grow aerobically at an iron concentration as low as 0.05 M without any of the known iron ionophores present. The growth rate increased between 0.05 and 2 M iron. Supplementation with the iron ligands ferrichrome and citrate resulted in optimal growth already at 0.05 M iron. Under certain conditions iron uptake preceded growth of cells by more than an hour. During logarithmic growth the rate of iron uptake matched the growth rate. The radioactive tracer method revealed a cellular iron content of 4 nmol/mg dry weight.After consumption of the iron in the medium cells continued to grow with high rate for 1–2 generations. The iron uptake activity was increased during iron starvation.     

Changes in Membrane Fatty Acid Composition and Δ12-Desaturase Activity during Growth of Acanthamoeba castellanii in Batch Culture

ABSTRACT. Growth of Acanthamoeba castellanii in batch culture at 30° C was associated with marked changes in cellular fatty acid composition. The largest change occurred in the linoleate to oleate ratio, which was maximal in early- to mid-exponential phase cultures but decreased approximately 10-fold as cells approached stationary phase. The higher degree of lipid unsaturation in young cultures was accentuated by a greater proportion of 20-carbon polyunsaturated fatty acids than in stationary phase cultures. The unsaturation index (average number of double bonds per fatty acid) was maximal in mid-exponential phase cultures after 24 hours growth. Incorporation of [1-¹⁴C]acetate into polyunsaturated fatty acids in short-term (2 hour) experiments was high in 12 and 24 hour old cultures, where linoleate and eicosadienoate accounted for up to 26% of total labelled fatty acids. Incorporation of [1-¹⁴CJacetate into these fatty acids was negligible in stationary phase cultures. These results were correlated with changes in the specific activity of the Δ12-desaturase. Δ12-Desaturase activity was greatest in microsomal membranes isolated from early- to mid-exponential phase cells, but declined by approximately 50% as cultures progressed towards stationary phase. Membrane fractionation studies revealed that although some differences in fatty acid composition between plasma-membrane, mitochondrial (enriched), and microsomal membrane fractions were evident, the large changes in lipid unsaturation in whole cells of A. castellanii could not be accounted for by differential development of particular subcellular membranes.     

Ingestion,fecundity, growth rates and culture of the harpacticoid copepod,Tisbe furcata,in the laboratory

Hydrobiologia - The ingestion, fecundity, growth rates and culture of theharpacticoid copepod, Tisbe furcata, fed different algalspecies at different cell densities were studied. It was foundthat...     

Effects of trialkyllead compounds on growth, respiration and ion transport in Escherichia coli K12

Triethyllead and tripropyllead cations affected growth, energy metabolism and ion transport in Escherichia coli K12. The tripropyllead compound was more liposoluble than the triethyl analogue and was also more effective in inhibiting cell growth and the oxygen uptake of both intact cells and membrane particles. Triethyllead acetate (5 microM) inhibited growth on non-fermentable carbon sources, such as glycerol and succinate, more markedly than on glucose. At higher concentrations, triethyllead caused significant inhibition of respiration rates of intact cells; the concentration giving 50% inhibition was 60 microM for glycerol-grown cells and 150 microM for glucose-grown cells. Oxidation of succinate by membrane particles was less sensitive to inhibition by the tripropyl- or triethyllead compounds than were the oxidations of DL-lactate or NADH. Triethyllead acetate [1.9 mumol (mg membrane protein)-1] inhibited the reduction by NADH of cytochromes; evidence for more than one site of inhibition in the respiratory chain was obtained. Membrane-bound ATPase activity was strongly inhibited by triethyllead acetate in the absence or presence of Cl-. The concentration of inhibitor giving 50% inhibition [0.02 mumol (mg membrane protein)-1] was about two orders of magnitude lower than that required for 50% inhibition of substrate oxidation rates in membranes. Triethyllead acetate (1 microM) induced swelling of spheroplasts in iso-osmotic solutions of either NH4Cl or NH4Br, presumably as a result of the mediation by the organolead compound of Cl-/OH- and Br-/OH- antiports across the cytoplasmic membrane. Similar exchanges of OH- for F-, NO3- or SO4(2)- or the uniport of H+ could not be demonstrated. Comparisons are drawn between the effects of trialkyllead compounds and those of the more widely studied trialkyltin compounds.