Characterisation of a siderophore from Acinetobacter calcoaceticus

Abstract The Gram-negatice bacterium Acinetobacter calcoaceticus was examined for production of siderophores and iron-repressible outer membrane proteins following growth in iron-restricted media. The iron chelator, 2,3-dihydroxybenzoic acid was identified in the culture supernatant bu ¹H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). A group of outer membrane proteins between 80 and 85 kDa were induced under iron restriction.     

Purification and properties of l-lactate dehydrogenase from Acinetobacter calcoaceticus

Abstract Membrane-bound l -lactate dehydrogenase has been purified almost to homogeneity from Acinetobacter calcoaceticus . The enzyme is an oligomeric protein of sub-unit M _r 40 000 containing non-covalently bound FMN as a prosthetic group. Purified l -lactate dehydrogenase has an apparent K _m of 83 μM for l -lactate but has no activity with, and is not inhibited by, d -lactate. The enzyme is strongly inhibited by HgCl₂, but other thiol reagents and metal-chelating compounds have little or no effect upon its activity.     

山梨糖脱氢酶在乙酸钙不动杆菌中的表达

目的:在乙酸钙不动杆菌Y2004中表达山梨糖脱氢酶。方法:将酮古龙酸菌山梨糖脱氢酶基因sdh以及从pWH1266质粒上扩增的复制原点ori先后酶切连接到pBBR1MCS2质粒上,构建pBBR1MCS2-ori-sdh穿梭质粒;再以pBBR1MCS2-ori-sdh/DH5α为供体菌、乙酸钙不动杆菌Y2004为受体菌、pRK2013/HB101为辅助菌进行三亲本接合转移;从氨苄青霉素和卡那霉素双抗平板上挑取转化子进行培养,通过菌落PCR和提取质粒复转筛选阳性克隆,再通过活性电泳和体外糖酸转化实验检测阳性克隆的山梨糖脱氢酶活性。结果:构建了pBBRMCS2-ori-sdh质粒并转入乙酸钙不动杆菌Y2004中,活性电泳和体外实验证实阳性克隆具有山梨糖脱氢酶活性。结论:实现了山梨糖脱氢酶在乙酸钙不动杆菌Y2004中的表达,为单菌糖酸转化的进一步研究奠定了基础。     

Novel carbenicillin-hydrolyzing β-lactamase (CARB-5) from Acinetobacter calcoaceticus var. anitratus

A strain of Acinetobacter calcoaceticus var. anitratus highly resistant to ticarcillin but susceptible to ticarcillin in combination with clavulanic acid (2 mg/l) was found to produce a constitutive beta-lactamase. This enzyme was periplasmic with a characteristic substrate profile of a carbenicillin-hydrolyzing enzyme. Enzyme inhibition was detected with antiserum (anti-CARB-3), pCMB, cloxacillin, clavulanic acid and sulbactam. This novel enzyme with a molecular mass of 28,000 resembles other plasmid-mediated carbenicillinases (CARB) but differs in its apparent isoelectric point estimated as 6.3 and has been designated CARB-5 on this basis.     

Cyclic AMP stimulates the protein tyrosine kinase activity of Acinetobacter calcoaceticus

The protein tyrosine kinase activity of Acinetobacter calcoaceticus was analyzed in vitro through the specific phosphorylation of an endogenous protein which is modified exclusively at tyrosine residues. A strong stimulation of this activity by cyclic AMP was observed. This finding represents the first example of a protein tyrosine kinase, in prokaryotes as well as in eukaryotes, whose functioning is cyclic nucleotide-dependent.     

Changes in NADP-isocitrate dehydrogenase isoenzyme levels in Acinetobacter calcoaceticus in response to acetate

Abstract During adaptation of Acinetobacter calcoaceticus to growth on acetate the specific activity of NADP-isocitrate dehydrogenase increased. This response is unique, as in other bacteria grown under the same conditions the activity of the enzyme decreases as a result of covalent phosphorylation. Moreover, A. calcoaceticus is also unusual in containing two distinct isoenzymes of NADP-isocitrate dehydrogenase. It has here been shown that the adaptation of A. calcoaceticus to acetate is accompanied by an increase in the relative proportion of the larger, allosteric, isoenzyme with a concomitant decrease in the level of the smaller, non-allosteric, isoenzyme.     

The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus LMD79.41

Quinoprotein glucose dehydrogenase (GDH; EC 1.1.99.17) was partially purified from cell-free extracts of Acinetobacter calcoaceticus LMD79.41. The enzyme oxidized monosaccharides (d-glucose, d-allose, 2-deoxy-d-glucose, d-galactose, d-mannose, d-xylose, d-ribose and l-arabinose) as well as disaccharides (d-lactose, d-maltose and d-cellobiose).Intact cells of A. calcoaceticus LMD79.41 also oxidized these monosaccharides, but not the disaccharides.The difference in substrate specificity can not be explained by impermeability of the outer membrane for disaccharides, since right-side-out membrane vesicles did not oxidize disaccharides either. Destruction of the cytoplasmic membrane strongly affected the catalytic properties of GDH. Not only did the affinity towards some monosaccharides change substantially, but disaccharides also became good substrates upon solubilization of the enzyme. Thus, at least in A. calcoaceticus LMD79.41, the oxidation of disaccharides by GDH can be considered as an in vitro ‘artefact’ caused by the removal of the enzyme from its natural environment.     

Struvite Precipitation Induced by a Novel Sulfate-Reducing Bacterium Acinetobacter calcoaceticus SRB4 Isolated from River Sediment

This article presents a study of struvite formation in liquid medium induced by the sulfate-reducing bacterium Acinetobacter calcoaceticus SRB4, a strain isolated from river sediment. We identified the bacterial strain A. calcoaceticus SRB4 and analyzed its micromorphology. The minerals formed were studied with an electroprobe microanalyzer, Fourier transform infrared spectroscopy, high-resolution transmission electron microscopy, selected-area electron diffraction, X-ray diffraction, thermogravimetry, differential thermogravimetry, and differential scanning calorimetry. Acinetobacter calcoaceticus SRB4 was found to induce struvite precipitation, whereas sterile control cultures did not. Many transparent stick-shaped struvite precipitates were distributed at the bottom of the conical flasks in the experimental group. Most bacteria were spherical and a large quantity of spherical struvite particles (less than 200 nm in diameter) adhered to the bacterial surface. An electron probe microanalysis showed that the precipitates contained C, O, P, Mg, and other elements. Fourier transformation infrared spectra showed that the precipitates contained crystalline water, NH₄⁺, and PO₄^3? groups. X-ray diffraction spectra showed that the precipitates were struvite crystals, with preferential orientation and lattice distortion. Thermogravimetry showed that the weight loss was caused by the evaporation of crystalline water at temperatures lower than 136°C and the release of ammonia from struvite at temperatures of 136–228.5°C. In this article, we discuss the possible mechanism of struvite formation and the possible role played by A. calcoaceticus SRB4. Our study extends our understanding of the phosphate biomineralization mechanism and should prove useful in recycling phosphorus in wastewater.     

Ecological role of Acinetobacter calcoaceticus GSN3 in natural biofilm formation and its advantages in bioremediation

This study assessed the role of a new Acinetobacter calcoaceticus strain, GSN3, with biofilm-forming and phenol-degrading abilities. Three biofilm reactors were spiked with activated sludge (R1), green fluorescent plasmid (GFP) tagged GSN3 (R2), and their combination (R3). More than 99% phenol removal was achieved during four?weeks in R3 while this efficiency was reached after two and four further operational weeks in R2 and R1, respectively. Confocal scanning electron microscopy revealed that GSN3-gfp strains appeared mostly in the deeper layers of the biofilm in R3. After four?weeks, almost 7.07?×?10⁷ more attached sludge cells were counted per carrier in R3 in comparison to R1. Additionally, the higher numbers of GSN3-gfp in R2 were unable to increase the efficiency as much as measured in R3. The presence of GSN3-gfp in R3 conveyed advantages, including enhancement of cell immobilization, population diversity, metabolic cooperation and ultimately treatment efficiency.     

Assimilatory nitrate reductase from Acinetobacter calcoaceticus

A soluble nitrate reductase from the bacterium Acinetobacter calcoaceticus grown on nitrate has been characterized. The reduction of nitrate to nitrite is mediated by an enzyme of 96000 molecular weight that can use as electron donors either viologen dyes chemically reduced with dithionite or enzymatically reduced with NAD(P)H, through specific diaphorases which utilize viologens as electron acceptors. Nitrate reductase activity is molybdenum-dependent as shown by tungstate antagonistic experiments and is sensitive to -SH reagents and metal chelators such as KCN.The enzyme synthesis is repressed by ammonia. Moreover, nitrate reductase activity undergoes a quick inactivation either by dithionite and temperature or by dithionite in the presence of small amounts of nitrate. Cyanate prevents this inactivating process and can restore the activity once the inactivation had occurred, thus suggesting that an interconversion mechanism may participate in the regulation of Acinetobacter nitrate reductase.Abbreviations EDTA ethylenediaminetetraacetate - BV benzyl viologen - MV methyl viologen - MW molecular weight - NEM N-ethylmaleimide - p-HMB p-hydroxymercuribenzoate - DCPIP 2,6-dichlorophenol-indophenol - FMN flavin mononucleotide - FAD flavin adenine dinucleotide - KCNO potassium cyanate     

Prevalence of IS(Aba1) in epidemiologically unrelated Acinetobacter baumannii clinical isolates

Seventy-five Acinetobacter baumannii strains belonging to different pulsetypes, plus one ceftazidime-susceptible strain, from a pulsetype in which all strains were resistant, were included in this study. The minimum inhibitory concentration of ceftazidime was determined by the microdilution method. The bla(ADC)-like gene, the IS(Aba1) element and the IS(Aba1) located in the bla(ADC)-like promoter were detected by PCR. The objective of the study was to determine the prevalence of IS(Aba1) in a collection of epidemiologically unrelated A. baumannii clinical isolates. The bla(ADC)-like gene was detected in 74 (97.3%) out of the 76 strains analysed. In these 74 strains, 51 (69%) were positive for the IS element and it was not detected in 23 (31%) strains. Among the A. baumannii strains containing the IS element, 40 (78.4%) had the IS element located in the promoter region of the bla(ADC)-like gene. In a high percentage of A. baumannii clinical isolates carrying the IS(Aba1), this is inserted into the promoter region of the bla(ADC)-like gene. In addition, two clinical isolates belonging to the same pulsetype, one with and one without the IS(Aba1), can be found in the clinical setting, suggesting the potential acquisition or loss of this genetic element in the hospital environment.     

Identification and nucleotide sequence of the Acinetobacter calcoaceticus encoded trpE gene

Summary The trpE gene from Acinetobacter calcoaceticus encoding the anthranilate synthase component I was cloned, identified by deletion analysis and sequenced. It encodes a predicted polypeptide of 497 amino acids with a calculated molecular weight of 55323. Its primary structure shows 49% identical amino acids with the enzyme from Clostridium thermocellum, 45% with that of Thermus thermophilus and only 35% with that of Escherichia coli. The codon usage of the trpE genes encoding the most homologous enzymes differs greatly indicating selection for amino acid maintainance. The homologies are clustered in the C-terminal 200 amino acids of the sequences indicating that this part is important for enzymic activity.     

Purification of a periplasmic insulin-cleaving proteinase from Acinetobacter calcoaceticus

Cells of Acinetobacter calcoaceticus contain a constitutive periplasmic metalloproteinase showing similar properties as the periplasmic metalloproteinase of Escherichia coli. The periplasmic proteinase of A. calcoaceticus was purified, starting from periplasm, by ammonium sulfate precipitation, hydrophobic interaction chromatography and chromatofocusing up to the homogeneity of the enzyme in SDS-electrophoresis with a yield of 6.7% and a purification factor of 417. The enzyme has a molecular mass of 108000 (gel filtration) or 112000 (native electrophoresis), and consists of four identical subunits with a molecular mass of 27 000 (SDS-electrophoresis). The purified enzyme degrades preferentially polypeptides such as glucagon and insulin. Larger proteins are accepted as substrates to a considerably lower extent. All tested synthetic substrates with trypsin, chymotrypsin, elastase and thermolysin specificity were not cleaved. Therefore, the described enzyme was designated “insulin-cleaving proteinase” (ICP).     

Enantiospecific Production of S-(-)-2-Hydroxypropiophenone Mediated by Benzoylformate Decarboxylase from Acinetobacter Calcoaceticus

Whole cells of Acinetobacter calcoaceticus, grown in a medium containing mandelate, converted benzoylformate and acetaldehyde into the acyloin compound 2-hydroxypropiophenone. The optical purity of the product was found to be greater than 98%. The absolute configuration of the biotransformation product at the carbinol carbon was found to be (S). The enzyme responsible for this bioconversion was confirmed as benzoylformate decarboxylase by the demonstration that the purified homogeneous enzyme catalysed the condensation reaction.     

The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii

ISAba1 was found in all widespread clones of Acinetobacter baumannii in the United Kingdom. All isolates studied had a blaOXA-51-like carbapenemase gene; some also had blaOXA-23-like and/or blaOXA-58-like. Among isolates with blaOXA-51-like as sole carbapenemase gene, only those with ISAba1 adjacent to blaOXA-51-like were carbapenem resistant. Minor differences in blaOXA-51-like sequence were observed in resistant and susceptible isolates. Isolates with blaOXA-23-like in addition were consistently resistant to carbapenems; in all of these ISAba1 lay upstream of blaOXA-23-like, but was not associated with blaOXA-51-like. These results suggest that ISAba1 is providing the promoter for blaOXA-51-like and, probably, for blaOXA-23-like.     

Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii

Abstract A new type II restriction endonuclease, named Ajo I, was detected in Acinetobacter johnsonii . The enzyme Ajo I, an isoschizomer of Pst I, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.     

Class I integrons containing a dhfrI trimethoprim resistance gene cassette in aquatic Acinetobacter spp

The presence of antibiotic resistance gene cassettes in class I integrons was investigated in 24 sulfamethoxazole-resistant and -sensitive Acinetobacter isolates derived from two Danish freshwater trout farms. Integrons were detected in five isolates from one of the fish farms, and their inserts were characterised by DNA sequencing. Each isolate contained a dhfrI gene cassette encoding resistance to trimethoprim and an open reading frame orfC of unknown function identical to the content of an integron previously found in a clinical enterobacterial isolate. Among the five isolates, at least two different strains were differentiated based on phenotypic tests and randomly amplified polymorphic DNA analysis. To our knowledge, this is the first report and characterisation of an integron in environmental bacteria.     

上呼吸道分离鲍曼不动杆菌1 型整合子的分离与鉴定

【目的】研究I型整合子的结构特征,探讨其与细菌多重耐药之间的相关性。【方法】收集2008年至2009年广州呼吸疾病研究所上呼吸道分离的187株鲍曼不动杆菌,应用K-B纸片扩散法检测耐药性,采用聚合酶链式反应进行I型整合子整合酶基因的检测;扩增整合子的可变区,应用DNA测序技术分析I型整合子基因结构。【结果】I型整合子的阳性率达53.4%。共七种1型整合子基因盒被鉴定,其中首次发现报道一种新的整合子(GenBank:HQ322622)。可变区主要编码氨基糖苷类药物的耐药基因。20种抗菌素耐药的结果均表明携带Ⅰ型整合子的鲍曼不动杆菌耐药率较不携带I型整合子的鲍曼不动杆菌的耐药率明显增高。整合子与鲍曼不动杆菌的多重耐药表型具有密切相关性。【结论】I类整合子相关耐药基因在本院临床分离鲍曼不动杆菌中分布较广泛。整合子在鲍曼不动杆菌耐药性的形成和播散中具有重要作用。     

Characterization and Fungal Inhibition Activity of Siderophore from Wheat Rhizosphere Associated Acinetobacter calcoaceticus Strain HIRFA32

Acinetobacter calcoaceticus HIRFA32 from wheat rhizosphere produced catecholate type of siderophore with optimum siderophore (ca. 92 % siderophore units) in succinic acid medium without FeSO₄ at 28 °C and 24 h of incubation. HPLC purified siderophore appeared as pale yellow crystals with molecular weight [M⁺¹] m/z 347.18 estimated by LCMS. The structure elucidated by ¹H NMR, ¹³C NMR, HMQC, HMBC, NOESY and decoupling studies, revealed that siderophore composed of 2,3-dihydroxybenzoic acid with hydroxyhistamine and threonine as amino acid subunits. In vitro study demonstrated siderophore mediated mycelium growth inhibition (ca. 46.87 ± 0.5 %) of Fusarium oxysporum. This study accounts to first report on biosynthesis of acinetobactin-like siderophore by the rhizospheric strain of A. calcoaceticus and its significance in inhibition of F. oxysporum.     

鲍曼不动杆菌对碳青霉烯类抗生素的耐药性及其基因的分析

目的研究23株鲍曼不动杆菌对碳青霉烯类抗生素的耐药情况及对耐药基因分析,为临床用药提供依据。方法用珠海迪尔DL-96鉴定系统进行细菌鉴定及K-B法进行药敏试验,用碳青霉烯酶4种基因的特异性引物对其进行聚合酶链反应(PCR)扩增和基因型分析,并通过网上GenBank进行比对以确定编码酶基因的类型。结果 23株鲍曼不动杆菌对哌拉西林/他唑巴坦、左旋氧氟沙星、丁胺卡那霉素、多黏菌素B的耐药率分别为80%、45%、30%、10%,对其他抗生素的耐药率均在90%以上。携带D类碳青霉烯酶OXA-23基因有18株(78%),携带OXA-51基因有15株(65%),OXA-24、OXA-58基因引物PCR扩增为阴性,随机各抽取3株OXA-23基因阳性株进行测序后通过在网上GenBank比对与OXA-23标准株99%同源,OXA-51基因阳性株与OXA-51标准株98%同源。结论耐碳青霉烯类抗生素的鲍曼不动杆菌对多黏菌素的耐药率最低,其次是丁胺卡那霉素,其中以携带OXA-23型碳青霉烯酶基因为主,应引起临床高度关注,防止在院内广泛传播。