Effect of Mg++ on the Activity of Vancomycin Against Escherichia coli

Mg⁺⁺ ions alleviate the inhibitory effect of vancomycin on Escherichia coli. This is not due to the formation of an antibacterial-inactive complex. It is suggested that vancomycin and Mg⁺⁺ compete for a receptor site, or sites, on (or in) the bacterial cell.     

The Effects of Environmental Conditions on the In Vitro Activity of Selected Antimicrobial Agents Against Escherichia coli

Various environmental conditions likely to be encountered at a nidus of infection were evaluated for their effect on selected classes of antimicrobial agents. The minimum inhibitory concentration (MIC) of several aminoglycosides (apramycin, kanamycin, gentamicin, tobramycin, amikacin), tetracycline, and chloramphenicol for five strains of E. coli were unchanged by temperature (35°–39.5°C), atmosphere (aerobic to anaerobic), pH > 7, NaCl concentration (up to 150 mM), zinc concentration (up to 50 mM), and manganese (up to 10 mM). However, the aminoglycoside MICs were increased up to fivefold at pH < 6.5. Magnesium and calcium ion concentrations >10 mM and ferric iron concentrations ≥10 mM increased aminoglycoside MICs from 3.66- to 8-fold. Tetracycline MICs were increased 1.2- to 6.5-fold when the concentration of magnesium or calcium was ≥10 mM. The results of this in vitro study might provide insight into the effects of local in vivo environmental conditions on several classes of antimicrobial agents. Received: 22 September 1997 / Accepted: 6 October 1997     

The Influence of Temperature on the Effect of Bacteriostatic Concentrations of Phenol Against Escherichia coli

S ummary . During early exponential growth of Escherichia coli in the absence of phenol there is a natural death rate at 20, 30, and 44° but at the optimum temperature around 37° there is little if any significant death. The influence of a rise in temperature from 20 to 44° is to decrease the generation time and at 44° the lower generation time compensates for a reduced generation index. The main effect of sub-bacteriostatic concentrations of phenol is to increase the generation time but at 30, 37 and 44° there is a significant reduction in the generation index at the higher concentrations resulting in a dynamic bacteriostasis. At 20° bacteriostasis is due mainly to a large generation time but there is a little growth and so bacteriostasis is essentially dynamic. There is also evidence to suggest that the effect of a particular concentration of phenol on the generation index is not merely influenced by the temperature but by the generation time under the particular set of conditions. If phenol is added to rapidly growing cultures of E. coli the effect of a rise in temperature is to reduce the concentration required for bacteriostasis but if it is added during the lag phase there is a maximum in the bacteriostatic concentration between 20 and 37°.     

Accumulation of Phosphatidic Acid Increases Vancomycin Resistance in Escherichia coli

In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane, preventing entry of toxic molecules such as antibiotics. Mutations in lptD, the beta-barrel component of the LPS transport and assembly machinery, compromise LPS assembly and result in increased antibiotic sensitivity. Here, we report rare vancomycin-resistant suppressors that improve barrier function of a subset of lptD mutations. We find that all seven suppressors analyzed mapped to the essential gene cdsA, which is responsible for the conversion of phosphatidic acid to CDP-diacylglycerol in phospholipid biosynthesis. These cdsA mutations cause a partial loss of function and, as expected, accumulate phosphatidic acid. We show that this suppression is not confined to mutations that cause defects in outer membrane biogenesis but rather that these cdsA mutations confer a general increase in vancomycin resistance, even in a wild-type cell. We use genetics and quadrupole time of flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) to show that accumulation of phosphatidic acid by means other than cdsA mutations also increases resistance to vancomycin. We suggest that increased levels of phosphatidic acid change the physical properties of the outer membrane to impede entry of vancomycin into the periplasm, hindering access to its target, an intermediate required for the synthesis of the peptidoglycan cell wall.     

Enhancing Antibacterial Activity Against Escherichia coli K-12 of Peptide Ib-AMP4 with Synthetic Analogues

A family of Ib-AMP4 peptide analogues was obtained by solid phase synthesis, modifying the net charge and hydrophobicity of C-terminal domain by replacing certain amino acidic residues by arginine and tryptophan. Additionally, disulfide bonds were eliminated by replacing the cysteine residues by methionine, which resulted in a decrease in the number of synthesis byproducts, and consequently diminished the subsequent purification steps. The obtained peptides were purified by RP-HPLC and their molecular mass was determined by MALDI-TOF mass spectrometry. The peptide analogues (IC₅₀ between 1 and 50 μM) presented a higher antibacterial activity against Escherichia coli K-12 than the native peptide (IC₅₀ > 100 μM). The hemolytic activity of the peptide with the highest antibacterial efficacy presented no degradation of erythrocytes for a concentration of 1 μM that corresponds to its IC₅₀ value. The results show that the synthesized peptides are good candidates for the treatment of diseases caused by E. coli.     

The Effect of Pyrophosphate Analogues on the Activity of the Inorganic Pyrophosphatase from Escherichia coli

The effect of inorganic pyrophosphate analogues on the enzymic activity of inorganic pyrophosphatase from E. coli was studied. Hypophosphoric and diphosphonic acids were shown to inhibit inorganic pyrophosphatase, whereas pyrophosphorous acid exerts almost no effect on the hydrolysis of inorganic pyrophosphate.     

Effect of Intracellular Concentration of Vitamin B6 on Tryptophanase Activity in Escherichia coli

The intracellular concentration of vitamin B6 (B6) of a wild type strain (WG1) of Escherichia coli B remained almost constant (0.25 to 0.35 nmol per mg cells) when different amounts of B6 were extracellularly added. However, B6-requiring mutants were strongly affected by extracellular B6.

Activities of tryptophanase in a B6-requiring mutant, strain WG3, grown under various conditions were measured. A clear correlation between intracellular B6 concentration and the ratio of holo-tryptophanase activity (i.e., holo-activity/total activity) was observed.

Tryptophanase of strain WG3 grown under B6-deficient conditions and the enzyme of strain WG1 were purified and their properties compared. The purified enzymes of both strains WG3 and WG1 showed similar characteristics, but a difference was observed in the antigen activities.     

壳聚糖对大肠杆菌的抑制作用规律及抗菌机理初探

考察了不同分子量壳聚糖对大肠杆菌的抑菌性能,利用壳聚糖的席夫碱反应对其氨基加以保护,探讨了壳聚糖对大肠杆菌的抗菌机理。研究结果表明:壳聚糖分子量越小,对大肠杆菌的抗菌作用越明显;壳聚糖对大肠杆菌的抑菌作用与其氨基的质子化有关。     

沼泽红假单胞菌生物合成银纳米粒子及其抗菌作用

近年来,利用沼泽红假单胞菌合成银纳米粒子作为一种可靠和环境友好的方法出现。主要利用沼泽红假单胞菌的细胞滤液来还原银离子。制备的纳米粒子用紫外可见光谱(UV-vis)、X射线衍射光谱(XRD)和透射电镜(TEM)进行表征。含有银粒子溶液的UV-vis光谱显示在420 nm-460 nm处出现银纳米粒子的吸收峰。TEM图像表明所形成的银纳米粒子的粒径范围为5 nm-20 nm。纳米粒子的XRD图谱证明产物为金属银。所制备的银纳米粒子对大肠杆菌和金黄色葡萄球菌作抑菌性试验。     

Evidence for Arylamine N-Acetyltransferase Activity in the Escherichia coli

N-Acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene as substrates were determined in isolates of the bacterium Escherichia coli. The N-acetyltransferase activity was determined by an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of E. coli isolates were found to be 0.67 ± 0.04 nmole/min/mg protein for 2-aminofluorene, and 0.46 ± 0.02 nmole/min/mg protein for p-aminobenzoic acid. The apparent K _m and V _max values obtained were 2.85 ± 0.65 mM and 7.51 ± 0.86 nmol/min/mg protein, respectively, for 2-aminofluorene, and 2.35 ± 0.39 mM and 9.43 ± 0.78 nmol/min/mg protein, respectively, for p-aminobenzoic acid. The optimal pH value for the enzyme activity was 7.0 for both substrates tested. The optimal temperature for enzyme activity was 37°C for both substrates. The N-acetyltransferase activity was inhibited by iodoacetamide: at 0.25 mM iodoacetamide, activity was reduced 50%, and at 1.0 mM, more than 90%. Among a series of divalent cations and salts, Cu²⁺ and Zn²⁺ were demonstrated to be the most potent inhibitors. This report is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in E. coli. Received: 29 April 1997 / Accepted: 2 July 1997     

A Critical Examination of Escherichia coli Esterase Activity

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances.The central question addressed in this paper is whether the glycosylated amino acids GlcNAc-Ser and GalNAc-Thr have been genetically encoded into proteins in vivo (1, 2). The reports for the incorporation of these two amino acids are unique from all other reports (3) that have incorporated unnatural amino acids using the recoded UAG codon and Methanococcus jannaschii orthogonal pairs in that these two amino acids required further processing by the host organism before incorporation (see Fig. 1). Here we posit that the primary barrier to their incorporation would appear to be the fact that the host organism used in the original reports, Escherichia coli, maintains very low levels of nonspecific esterase activity. In fact, the original reports used citations from mammalian biology to substantiate the nonspecific esterase mechanism (see below).Open in a separate windowFIGURE 1.Proposed product of an esterase with GlcNAc-Ser and other esterase substrates discussed in this study.E. coli is likely the most thoroughly studied microorganism. This is especially true in regard to carbohydrate and amino acid uptake and utilization (4). Therefore, it should not be surprising that it has long been known that esterified carbon sources are not metabolized by E. coli in standard assays used to probe for microorganism lipase and esterase activity (5). Such results and our current analysis underscore the limitations of the reports that triacetyl O-linked glycosylated amino acids (GlcNAc-Ser and GalNAc-Thr) were deacetylated in E. coli by endogenous “nonspecific” esterases. The deacetylated amino acids were then believed to have been genetically encoded into full-length proteins in vivo (1, 2).In these previous studies the glycosylated amino acids were provided to the growth media as their tetraacetate analogs, and it was construed from the mass spectra and lectin binding assays that the ester groups of the saccharide had all been hydrolyzed. The notion that E. coli rapidly hydrolyzes a simple ester is not easily reconciled with what is commonly observed when the ester functional group is introduced into cultures of E. coli. For example, we were prompted by reports that claimed to have harvested β-hydroxy esters from E. coli (6). There was nothing in such a report to indicate that the E. coli strain used had undergone a drastic genetic modification beyond the introduction of one enzyme derived from yeast. The enzyme from yeast was expressed in E. coli to asymmetrically reduce β-keto esters to the corresponding β-hydroxy esters. The reduction was accomplished in 87% yield and was performed in whole cells. It stands to reason that such a report having claimed to extract significant amounts of an esterified product would not be possible if E. coli maintained even moderate levels of nonspecific esterase activity. The fact that E. coli maintains low levels of endogenous esterases and lipases has been quite pivotal for a number of studies that have used this organism as the host to express esterase genes in vivo (see below).Nonspecific esterase activity is common in eukaryotic organisms, for example, our ability to hydrolyze triacylglycerides to access an important energy source, but this stands in stark contrast to E. coli where it is possible to directly extract O-acetylated oligosaccharides (7) and other simple esters (6) in high yields. These reports are consistent with the observation that UDP-2,3-diacylglucosamine accumulates in E. coli when genes from lipid biosynthesis are deleted (8). E. coli is also the preferred host for evaluating esterase and lipase activity when screening genes from cultured and uncultured organisms (9, 10). Screening for lipase activity from various microorganisms is often performed on tributyrin agar plates (11). The results are typically the same as for triacetin, and it is repeatedly observed that E. coli does not naturally grow on triesters of glycerol (12, 13). These and many other similar esterase screens (14) would not have been feasible if E. coli produced even moderate levels of a lipase or nonspecific esterase.In the present article we use a combination of our current findings and a thorough review of the relevant literature to conclude that E. coli may not maintain sufficient levels of nonspecific esterase activity to permit the in vivo incorporation of the glycosylated amino acids by the mechanism reported (Fig. 1). Our conclusion is further supported by isothermal calorimetry measurements of Zhang et al. (1) original enzyme showing it retains considerable wild-type activity. We also show that the amino acid GlcNAc-Ser appears to be metabolized in E. coli.