Antimicrobial dendrimer active against Escherichia coli biofilms

We have investigated the ability of a previously reported antimicrobial peptide dendrimer (RW)_4D to inactivate Escherichia coli RP437 in planktonic culture and in biofilms. The results show that the dendrimer inhibits bacterial growth in both planktonic and biofilm states. Live/Dead staining assays reveal that most bacteria in a preformed biofilm lose viability after treatment with this peptide. This result is in marked contrast to most existing reports that antimicrobial peptides are ineffective against mature bacterial biofilms.     

亮氨酸脱氢酶与葡萄糖脱氢酶高效共表达制备L-叔亮氨酸北大核心CSCD

【目的】通过不同双基因共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶基因在大肠杆菌中表达影响的研究,获得具有高辅酶再生效率的双酶共表达重组生物催化剂,实现L-叔亮氨酸"一锅法"高效不对称合成。【方法】以来自于蜡状芽孢杆菌(Bacillus cereus)的亮氨酸脱氢酶(LDH)和来自芽孢菌属(Bacillus sp.)的葡萄糖脱氢酶(GDH)为模板,考察单质粒共表达,双质粒共表达和融合表达等3种共表达策略对重组细胞中亮氨酸脱氢酶和葡萄糖脱氢酶活的影响,比较不同酶活比例和不同催化剂形式对三甲基丙酮酸不对称还原制备L-叔亮氨酸效率的影响。【结果】研究发现不同共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶的影响存在明显差异。亮氨酸脱氢酶在不同策略下均能够正常表达,而葡萄糖脱氢酶在融合表达时没有活力,当C端含有组氨酸标签时,表达蛋白活性低。通过表达优化,获得3株亮氨酸脱氢酶和葡萄糖脱氢酶高效表达且具有不同酶活比例的重组菌。比较粗酶液和全细胞形式下的催化效率,发现酶活比例及催化剂形式对不对称还原反应效率具有重要影响。确定单质粒串联表达C端不含His标签重组菌E.coli BL21/p ET28a-L-SD-AS-G为最佳催化剂,以粗酶液进行转化时,完全转化0.5 mol/L底物所需菌体量为15 g/L,辅酶量为0.1 mmol/L。【结论】采用单质粒共表达策略,成功构建出1株具有较高亮氨酸脱氢酶和葡萄糖脱氢酶活性的重组菌,实现高效催化TMP合成L-Tle。     

High-level expression of an altered cDNA encoding human isovaleryl-CoA dehydrogenase in Escherichia coli

Isovaleryl-CoA dehydrogenase (IVD) catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway. The cDNA encoding the mature human IVD polypeptide was cloned in a prokaryotic expression vector, but the level of expression in Escherichia coli was extremely low and attempts to purify the enzyme to homogeneity were unsuccessful. To enhance expression, the nucleotide sequence of 22 codons within the 111-bp region at the 5′-end of the cDNA was altered to accommodate E. coli codon usage without altering the amino-acid coding sequence. The altered IVD cDNA was synthesized by PCR, using a primer containing the desired modifications. Following overnight induction of the E. coli transformed with this cDNA, the enzyme was purified to homogeneity using diethylaminoethyl agarose and high-pressure ceramic hydroxyapatite resins. IVD activity was increased 165-fold in the crude extract of cells containing the modified cDNA, as compared to that containing the wild-type cDNA.     

Caffeine-death in Escherichia coli

Summary Growth of a culture of E. coli strain B or 15 in medium containing caffeine resulted in the accumulation of inviable cells in the population. A caffeine concentration of 8 mM caused the death of between 30% and 50% of the cells in 12 independent populations grown for 15 generations or more. The thymine dimer excision-defective strains Bs-1, Bs-8 and Bs-12 and the exr ^– mutant Bs-2 were resistant to this lethal effect. The reckless, hcr ⁺ mutant Bs-11 was more sensitive than the parental B strain. Although 100mM caffeine did not impair DNA synthesis in vitro, concentrations of the drug 8 mM caused a significant decline in DNA synthesis in vivo in E. coli B cells. From the fit of an experimental growth curve to an algebraic model of growth in which a proportion of cells are inactivated at each replication it is suggested that caffeine does not affect the replication rate of the viable cells. The observed impairment of DNA synthesis in vivo is equated with this cell death (caffeine-death). For E. coli 15 or B, 8 mM caffeine induced caffeine-death at a rate of 18% per cell generation. Caffeine-resistant mutants of E. coli B and E. coli 15 were isolated. Of those studied in detail a substantial proportion proved to be U.V. and X-ray sensitive and excision-defective. Others were more U.V. and X-ray resistant than strain B. Yet another class proved highly unstable. A chromosome breakage model of caffeine-death implicating enzymes of the excision-repair process is discussed.     

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K_M was 42 mM, and V_max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.     

Morphogenesis of Escherichia coli

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

大肠杆菌突变株高密度生长的多因素分析

[背景]pBHR68是表达聚-3-羟基丁酸酯(Poly-3-Hydroxybutyrate,PHB)合成基因簇的高拷贝质粒,大肠杆菌K-12突变菌株S17-3在携带该质粒时生长密度高,耐低pH且在低pH条件下生长时高产可拉酸(Colanic Acid,CA).[目的]系统探究与菌种(大肠杆菌S17-3)及质粒(pBHR...     

The cyclic AMP receptor of Escherichia coli: Immunological studies in extracts of Escherichia coli and other organisms

An antiserum specific for the cyclic adenosine 3′,5′-monophosphate receptor from Escherichia coli has been employed to detect the presence of a similar protein in cellular extracts of a number of diverse organisms. In Ouchterlony double-diffusion experiments cellular extracts from Photobacterium fisheri, Aerobacter aerogenes, Proteus mirabilis, and Salmonella typhimurium all showed precipitin bands with E. coli cyclic AMP receptor-antiserum. The extract from Caulobacter crescentus exhibited slight cross-reactivity. Similar results were obtained with an immuno-precipitation assay used to quantitate the amount of cyclic AMP receptor-like protein present. Extracts from a variety of organisms were found to bind cyclic AMP when the usual (NH₄)₂SO₄ precipitation assay for cyclic AMP receptor was employed. Only the extract from Methanosarcina barkeri was inactive. Some extracts prepared from E. coli grown on Luria broth were observed to have no cyclic AMP binding activity. Antiserum was used to determine the presence of cyclic AMP receptor in these inactive extracts. These preparations usually regain binding activity on standing at 4°C for 2–3 days.     

Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

Background  

The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

Mutagenic DNA repair in Escherichia coli

Summary The photoreversibility of UV-induced mutations to Trp⁺ in strain Escherichia coli WP2 uvrA trp (unable to excise pyrimidine dimers) was lost at different rates during incubation in different media. In Casamino acids medium after a short initial lag, photoreversibility was lost over about one generation time; in minimal medium with tryptophan, photoreversibility persisted for more than two generations; in Casamino acids medium with pantoyl lactone photoreversibility was lost extremely slowly. The rate of loss of photoreversibility was unaffected by UV dose in either Casamino acids medium or in minimal medium. The same eventual number of induced mutants was obtained when cells were incubated for two generations in any of the three media before being transferred to selective plates supplemented with Casamino acids. Thus in each the proportion of cells capable of giving rise to a mutant was the same and only the rate at which these cells did so during post-irradiation growth varied, suggesting that there might be a specific fraction of pyrimidine dimers at a given site capable of initiating a mutagenic repair event, and that the size of this fraction is dose dependent. Segregation experiments have shown that error-prone repair appears to occur once only and is not repeated in subsequent replication cycles, in contrast to (presumed error-free) recombination repair.The results are discussed in the light of current models of UV mutagenesis.     

Escherichia coli and its chromosome

The Escherichia coli chromosome is a circular DNA molecule that is approximately 1000 times compacted in the living cell, where it occupies approximately 15% of the cellular volume. The genome is organized in a way that facilitates chromosome maintenance and processing. Despite huge efforts, until recently little has been known about how the chromosome is organized within cells, where replication takes place, and how DNA is segregated before cell division. New techniques for labeling genetic loci and molecular machines are allowing the simultaneous tracking of genetic loci and such machines in living cells over time. These studies reveal remarkable organization, yet a highly dynamic flux of genetic loci and macromolecules. It seems likely that the cellular positioning of chromosomal loci is the outcome of the formation of two chromosome arms (replichores) by replication, followed by sequential chromosome segregation, rather than from the presence of cellular positioning markers.     

Mutagenic DNA repair in Escherichia coli

Summary Escherichia coli K12 Hfr H Tsx^s Str^s and F^- Pro^- Tsx^r His^- Arg^- Str^r bacteria were conjugated in the absence of arginine with or without glucose. The efficiency of conjugation, measured by the frequency of Pro⁺ and His⁺ recombinants was not affected. Arginine starvation alone did not affect the tsx ^s gene expression which occurred in all the zygotes which had received the gene. In contrast, argine and glucose starvation allows tsx ^s expression only in those zygotes in which the donor gene had been integrated in the genome. As the glucose starvation brings on a destabilization of the messenger RNA synthesized by the F^- cells in absence of arginine, the results can be interpreted as follows: the transferred tsx ^s genes are transitorily expressed in all the zygotes at the unintegrated state. After this transient period, only those genes integrated in the chromosomes of the zygotes continue to be expressed.     

Ribosomal protein modification in Escherichia coli

Summary A mutant of Escherichia coli K12 has been isolated which shows an alteration in the ribosomal protein S18. Genetic analyses have revealed that the mutation causing this alteration maps at 99.3 min of the E. coli genetic map, between dnaC and deo. This indicated that the mutation has occurred in a gene different from the structural gene for this protein which has been located at 94 min. From the N-terminal amino acid sequence analysis it is concluded that the mutation has resulted in loss of the N-terminal acetyl group of this protein. The gene which is affected in this mutant is termed rimI that most likely specifies an enzyme acetylating the N-terminal alanine of protein S18. The mutation does not affect the acetylation of two other ribosomal proteins, S5 and L12, both of which are known to be acetylated in wild-type E. coli K12.     

Ribosomal protein modification in Escherichia coli

Summary Among mutants of E. coli selected for temperaturesensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the acetylase activity specific for protein L12 was negligible, when assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL that codes for an acetylating enzyme specific for ribosomal protein L12.Previous paper in this series is Isono and Isono (1980)     

Properties of γ-aminobutyraldehyde dehydrogenase from Escherichia coli

γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an M_r of 195 000±10 000 in its dimeric form with an M_r of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. K_m values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD⁺, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

Mutations affecting the reduced nicotinamide adenine dinucleotide dehydrogenase complex of Escherichia coli

A strain carrying a point mutation affecting the NADH dehydrogenase complex of Escherichia coli has been isolated and its properties examined. The gene carrying the mutation (designated ndh) was located on the E. coli chromosome at about minute 23 and was shown to be cotransducible with the pyrC gene. Strains carrying the ndh^? allele were found to be unable to grow on mannitol and to grow very poorly on glucose unless the medium was supplemented with succinate, acetate or casamino acids.The following properties of strains carrying the ndh^? allele were established which suggest that the mutation affects the NADH dehydrogenase complex but apparently not the primary dehydrogenase. Membrane preparations possess normal to elevated levels of d-lactate oxidase and succinate oxidase activities but NADH oxidase is absent. NADH is unable to reduce ubiquinone in the aerobic steady state and reduces cytochrome b very slowly when the membranes become anaerobic. NADH dehydrogenase, measured as NADH-dichlorophenolindophenol reductase is reduced but not absent. NADH oxidase is stimulated by menadione although not by Q-3 or MK-1 and in the presence of menadione, cytochrome b is reduced normally by NADH.Further mutants affected in NADH oxidase were isolated using a screening procedure based on the growth characteristics of the original ndh^? strain. The mutations carried by these strains were all cotransducible with the pyrC gene and the biochemical properties of the additional mutants were similar to those of the original mutant.The properties of the group of ndh^? mutants established so far suggest that they are affected in the transfer of reducing equivalents from the NADH dehydrogenase complex to ubiquinone.     

Analysis of rpsD mutations in Escherichia coli

Summary A certain proportion of protein S7 exists in an altered form in E. coli rpsD (S4) mutants. Depending on the type of S4 mutation involved, two different forms of the altered S7 can be distinguished. The unusual form is longer than normal S7 by about 500 daltons due to extra material at the carboxyl end of the protein. It is suggested that a mutationally altered S4 might lower the efficiency of termination during translation of the messenger for S7. This results in an increased frequency of translational read-through, which gives the observed longer forms of S7. Data are interpreted to mean that one class of S4 mutants might suppress UGA and UAG whereas another class only suppresses UGA.