重组细菌多药外排泵AcrAB-TolC的转运功能和动态组装

【背景】多药外排泵多以膜蛋白复合体形式存在,是导致细菌耐药性的重要原因。外排泵的转运功能和组装过程对于细菌耐药性和药物研发具有重要意义。【目的】以多药外排泵耐药结节细胞分化家族(resistance-nodulation-division family, RND)的重要成员AcrAB-TolC复合体为对象,研究其转运活性和体外组装特性。【方法】基于大肠杆菌AcrAB-TolC复合体基因序列,分别构建含有acrA、acrB、tolC基因的重组质粒,表达和纯化复合体各亚基,利用荧光光谱、等温滴定量热法(isothermal titration calorimetry,ITC)等技术分析复合体及亚基的转运功能、亚基与底物的相互作用,以及亚基间的相互作用和动态装配。【结果】实现了AcrAB-TolC复合体各组分的表达和纯化(纯度>98%),证实表达有各组分的活细胞提高了对于溴化乙锭(ethidium bromide,EB)的转运活性,并发现群体感应效应信号分子N-hexanoyl-L-homoserine lactone (C6-HSL)能够抑制AcrB、TolC对于EB的转运活性。ITC结果进一步证实了C6-HSL与AcrB、TolC的相互作用。ITC结果还显示AcrA分别与AcrB、TolC之间存在明显的相互作用,而AcrB与TolC之间无明显的相互作用。在体外装配实验中观测到AcrAB-TolC亚基的单分子荧光强度随时间增加,证实了复合体亚基在膜上的动态组装过程。【结论】实现了AcrAB-TolC外排泵及亚基的表达和纯化,证实了AcrAB-TolC对底物的转运活性及与底物的相互作用,观察到AcrAB-TolC的动态组装过程。以上结果为研究多药外排泵导致的细菌耐药性及抗菌策略具有重要意义。     

Characterization of chloramphenicol resistance inLactobacillus plantarum caTC2

A strain ofLactobacillus plantarum caTC2R isolated from a meat source was resistant to chloramphenicol (30 g/ml). Resistance was mediated through an inducible chloramphenicol acetyltransferase. Plasmid analysis of this strain showed three plasmids, of which the 8.5-kb plasmid apparently encodes the gene for chloramphenicol resistance. This plasmid was lost at high frequency (25%) when theLactobacillus was subcultured at a higher than optimal temperature (40°C).     

Cis effect oflacZ sequences in transgenic mice

Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving theEscherichia coli -galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression inHMG-lacZ transgenic mice. The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene. Two lines of doubleHMG-lacZ andHMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites. These mice expressed both reporter genes, but exclusively in the testes. These results suggest that thelacZ sequence might interfere negatively with the expression of the adjacentHMG-cat transgene.     

Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes

Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.     

Production of isoamyl acetate in<Emphasis Type="Italic"> ackA-pta</Emphasis> and/or<Emphasis Type="Italic"> ldh</Emphasis> mutants of<Emphasis Type="Italic"> Escherichia coli</Emphasis> with overexpression of yeast<Emphasis Type="Italic"> ATF2</Emphasis>

The gene coding for alcohol acetyltransferase (ATF2), which catalyzes the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA), was cloned from Saccharomyces cerevisiae and expressed in Escherichia coli. This genetically engineered strain of E. coli produced the ester isoamyl acetate when isoamyl alcohol was added externally to the cell culture medium. Various competing pathways at the acetyl-CoA node were inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Several strains with deletions in the ackA-pta and/or ldh pathways and bearing the ATF2 on a high-copy-number plasmid were constructed and studied. Compared to the wild-type, ackA-pta and nuo mutants produced higher amounts of ester and an ackA-pta-ldh-nuo mutant lower amounts. Isoamyl acetate production correlated well with intracellular coenzyme A (CoA) and acetyl-CoA levels. The ackA-pta-nuo mutant had the highest intracellular CoA/acetyl-CoA level and hence produced the highest amount of ester (1.75 mM) during the growth phase under oxic conditions and during the production phase under anoxic conditions.     

Increased Resistance to Acriflavine by Amplification of the acrA Gene of Escherichia coli K-12

The wild-type acrA⁺ gene of Escherichia coli K-12, cloned intoplasmid pAF1, was expressed as resistance to acriflavine (AF)in AF-sensitive acrA mutant cells (N43). When acrA⁺ genes wereamplified by treatment of cultures with chloramphenicol (50µg/ml), cells expressed much higher resistance to AF thanthat of the wild-type strain (N90). (Received November 22, 1989; Accepted July 7, 1990)     

2-Bromoethanesulfonate: A selective agent for isolating resistantMethanosarcina mutants

Sodium 2-bromoethanesulfonate (BES), a structural analog of 2-mercaptoethanesulfonate (coenzyme M), inhibited methanogenesis and growth ofMethanosarcina strain 227 in the presence of H₂/CO₂, methanol, or acetate. A single exposure to 24 M BES was sufficient to produce cultures resistant to 240 M BES. Wild-type cultures inhibited by 200 M BES (or less) resumed growth and methane production when coenzyme M (coM) was added to the culture medium. Cultures incubated one week or longer with 200 M BES (or less) spontaneously resumed growth and methanogenesis in the presence of H₂/CO₂, methanol, or acetate without added coM. BES resistance was heritable and not the result of inactivation or decomposition of BES. BES resistance acquired on one methanogenic substrate was retained when cells were grown on a different methanogenic substrate. However, BES resistance did not confer multiple resistance to other halomethane compounds such as chloroform, 2-bromoethanol, 2-bromopropionic acid, and chloramphenicol. BES resistance varied in two other genera of methanogens tested. One strain ofMethanospirillum hungatei was very sensitive to BES, and no resistant mutants were demonstrated. One strain ofMethanobacterium formicicum, however, was resistant to 200 M BES without any known prior exposure to BES.     

Detection of acetyl coenzyme A as an early CO2 assimilation intermediate in Methanobacterium

The pivotal role of acetyl coenzyme A in CO₂ assimilation by autotrophic methanogenic bacteria has been demonstrated by pulse-labelling of growing Methanobacterium thermoautotrophicum with ¹⁴CO₂. After very short incubation with ¹⁴CO₂ (1.5 s) approximately 1% of label incorporated into the soluble cell fraction was contained in acetyl coenzyme A. The percentage distribution of ¹⁴C within acetyl CoA markedly decreased with time, which is indicative for acetyl CoA being an immediate ¹⁴CO₂ fixation product. Label in the acetate molecule first appeared in the carboxyl carbon, but the methyl carbon became equally labelled within only 10 s. The acetyl CoA was compared with authentic material by various criterions and its cellular concentration was determined to be 52 M. This small cellular pool size of acetyl CoA as compared to e.g. alanine (6.4 mM) provides an explanation for the observed labelling kinetics. The data are fully consistent with autotrophic carbon assimilation via a total synthesis of acetyl coenzyme A from 2 CO₂.Dedicated to Professor Dr. Gerhart Drews on occasion of his 60th birthday     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

The Bacillus subtilis small cytoplasmic RNA gene and ''dnaX'' map near the chromosomal replication origin.

Summary TheBacillus subtilis small cytoplasmic RNA (scRNA) has an important, although not yet defined function in protein biosynthesis. Here we describe the mapping of the single copy scRNA gene and the flanking homolog todnaZX ofEscherichia coli, termed dnaX. The scRNA gene region of aB. subtilis wild-type strain was marked with acat gene and mapped by scoring chromosomal co-transformation rates of various mutant strains to chloramphenicol resistance and loss of the mutant phenotypes, respectively. This analysis, together with anEcoRI map comparison, places the scRNA gene anddnaX in the vicinity ofrecM near the replication origin region ofB. subtilis.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Concurrence of cat and tet Genes in Multiple Antibiotic-Resistant Bacteria Isolated from a Sea Cucumber and Sea Urchin Mariculture Farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.     

First Emergence of acrAB and oqxAB Mediated Tigecycline Resistance in Clinical Isolates of Klebsiella pneumoniae Pre-Dating the Use of Tigecycline in a Chinese Hospital

Tigecycline is one of the few therapeutic options for treating infections caused by some multi-drug resistant pathogens, such as Klebsiella pneumoniae. However, tigecycline-resistant K. pneumoniae has been discovered recently in China. From 2009 to 2013, nine tigecycline-resistant K. pneumoniae isolates were identified in our hospital. Six of nine strains were identified before using tigecycline. To investigate the efflux-mediated resistance mechanisms of K. pneumoniae, the expression of efflux pump genes (acrA, acrB, tolC, oqxA and oqxB) and pump regulators (acrR, marA, soxS, rarA, rob and ramA) were examined by real-time RT-PCR. Molecular typing of the tigecycline resistant strains was performed. ST11 was the predominant clone of K. pneumoniae strains, while ST1414 and ST1415 were novel STs. Efflux pump inhibitor (EPI)-carbonyl cyanide chlorophenylhydrazone (CCCP) was able to reverse the resistance patterns of 5 resistant K. pneumoniae strains. In comparison with strain A111, a tigecycline-susceptible strain (negative control), we found that the expression levels of efflux pump genes and pump regulators were higher in a majority of resistant strains. Higher expression levels of regulators rarA (2.41-fold, 9.55-fold, 28.44-fold and 18.31-fold, respectively) and pump gene oqxB (3.87-fold, 31.96-fold, 50.61-fold and 29.45-fold, respectively) were observed in four tigecycline resistant strains (A363, A361, A368, A373, respectively). Increased expression of acrB was associated with ramA and marA expression. To our knowledge, studies on tigecycline resistance mechanism in K. pneumoniae are limited especially in China. In our study, we found that both efflux pump AcrAB-TolC and OqxAB contributed to tigecycline resistance in K. pneumoniae isolates.     

In-silico interaction studies suggest RND efflux pump mediates polymyxin resistance in Acinetobacter baumannii

Bacterial efflux pumps have emerged as antibiotic resistance determinants and confers multi-drug resistance to a broad range of antimicrobials as well as non-antibiotic substances. A study about translocation of antibiotic molecules through the efflux transporter, will contribute in determining substrate specificity. In the present study, we have explored RND family efflux pump extensively found in Acinetobacter baumannii i.e. AdeABC. Besides, another well studied RND efflux pump, AcrAB-TolC together with a non-RND efflux pump, NorM was investigated for comparative analysis. We employed a series of computational techniques ranging from molecular docking to binding free energy estimation and molecular dynamics simulations to determine the binding affinity for different classes of drugs, namely aminoglycosides, polymyxins, β-lactams, tetracyclines, glycylcyclines, quinolones and metronidazole with AdeB, AcrB, and NorM efflux proteins. Our results revealed that class polymyxins has the highest binding affinity with the RND efflux pumps i.e. AcrAB-TolC and AdeABC as well as non-RND efflux pump, NorM. The experimental validation study demonstrated bigger zone of inhibition in presence of efflux pump inhibitor than polymyxin alone thus unveiling its specificity toward efflux pump. The reported experimental data comprising of minimum inhibitory concentration of antibiotics toward these efflux pumps also support our finding based on in silico approach. To recapitulate the outcome, polymyxins shows maximum specificity toward RND as well as non-RND efflux pump and may unlatch the way to rationally develop new potential antibacterial agents as well as efflux pump inhibitors in order to combat resistance.     

Contributions of binding and catalytic rate constants to evolutionary modifications inKm of NADH for muscle-type (M4) lactate dehydrogenases

Summary The apparent Michaelis constant (K _m) of NADH for muscle-type (M₄ isozyme) lactate dehydrogenases (LDHs) is highest, at any given temperature of measurement, for LDHs of cold-adapted vertebrates (Table 1). However, these interspecific differences in theK _m of NADH are not due to variations in LDH-NADH binding affinity. Rather, theK _m differences result entirely from interspecific variation in the substrate turnover constant (k _cat) (Fig. 1; Table 2). This follows from the fact that theK _m of NADH is equal tok _cat divided by the on constant for NADH binding to LDH,k ₁, so that interspecific differences ink _cat, combined with identical values fork ₁ among different LDH reactions, make the magnitude of theK _m of NADH a function of substrate turnover number. The temperature dependence of theK _m of NADH for a single LDH homologue is the net result of temperature dependence of bothk _cat andk ₁ (Figs. 3 and 4). Temperature independentK _m values can result from simultaneous, and algebraically offsetting, increases ink _cat andk ₁ with rising temperature. Salt-induced changes in theK _m of NADH also may be due to simultaneous perturbation of bothk _cat andk ₁ (Table 3). These findings are discussed from the standpoint of the evolution of LDH kinetic properties, particularly the interspecific conservation of catalytic and regulatory functions, in differently-adapted species.     

Controlled gene expression utilising Lambda phage regulatory signals in a cyanobacterium host

Summary This study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A. nidulans R2 derivative strain. An operator-promoter region and the temperature-sensitive repressor gene cI857 of bacteriophage Lambda were employed. Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host. The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift. We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor. The systems described should allow controlled expression of adventitious genes in the cyanobacterial host.Abbreviations AP^r ampicillin resistance - Cm^r chloramphenicol resistance - CmActase chloramphenicol acetyltransferase - Km^r Kanamycine resistance - [ ] indicates plasmid carrier state     

The Utilization of Choline and Acetyl Coenzyme A for the Synthesis of Acetylcholine

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high-affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high-affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high-affinity transport and acetylation of choline are kinetically coupled.     

Susceptibility,resistance development,and synergy of antimicrobial combinations againstClostridium difficile

Studies of susceptibility, development of resistance, and synergy were performed with 32 antimicrobics againstClostridium difficile strains. A microtiter technique was used to demonstrate the in vitro activities of 40C. difficile isolates. Rifampin inhibited 73% of the strains at a concentration of 0.06 g/ml, and metronidazole inhibited 90% at 0.5 g/ml. Penicillin, vancomycin, cephaloridine, and chloramphenicol were also active. Development of resistance to penicillin or chloramphenicol was not found, but vancomycin, methicillin, and minocycline showed development of low levels of resistance. Rifampin in combination with erythromycin, methicillin, or penicillin was synergistic against more than 90% of theC. difficile isolates. Rifampin-ampicillin was synergistic against 85%, and vancomycin-metronidazole was synergistic against 68% of the isolates.