Genetic Organization of Genes Encoding Phenol Hydroxylase, Benzoate 1,2-Dioxygenase Alpha Subunit and Its Regulatory Proteins in Acinetobacter calcoaceticus PHEA-2

Acinetobacter calcoaceticus PHEA-2 is a phenol-degrading bacterium isolated from the wastewater from an oil refinery. A 10-kb XhoI fragment consisting of nine complete Open Reading Frames (ORFs) and one partial ORF was screened from a lambda library of PHEA-2 by Southern hybridization. The sequence analyses revealed that ORF2–ORF7, designated mphKLMNOP, are homologous to dmpKLMNOP of Pseudomonas sp. CF600 and mopKLMNOP of Acinetobacter calcoaceticus NCIB8250, sharing 38%–72% and 58.5%–93.5% respectively. The products encoded by dmp and mop genes convert phenol to catechol. The mph-operon and downstream ORFs, ORF9 and ORF10, sharing high identities to benM and benA, which encode ben-operon regulatory protein and benzoate 1,2-dioxygenase alpha subunit respectively, are separated by ORF8, whose function is unknown. The organization of the mph and ben operons is different from that described previously. Received: 8 April 2002 / Accepted: 8 May 2002     

Genome sequence of Acinetobacter calcoaceticus PHEA-2, isolated from industry wastewater

Genome analysis of Acinetobacter calcoaceticus PHEA-2 was undertaken because of the importance of this bacterium for bioremediation of phenol-polluted water and because of the close phylogenetic relationship of this species with the human pathogen Acinetobacter baumannii. To our knowledge, this is the first strain of A. calcoaceticus whose genome has been sequenced.     

Genetic organization of genes encoding phenol hydroxylase,benzoate 1,2-dioxygenase alpha subunit and its regulatory proteins in Acinetobacter calcoaceticus PHEA-2

Acinetobacter calcoaceticus PHEA-2 is a phenol-degrading bacterium isolated from the wastewater from an oil refinery. A 10-kb XhoI fragment consisting of nine complete Open Reading Frames (ORFs) and one partial ORF was screened from a lambda library of PHEA-2 by Southern hybridization. The sequence analyses revealed that ORF2-ORF7, designated mphKLMNOP, are homologous to dmpKLMNOP of Pseudomonas sp. CF600 and mopKLMNOP of Acinetobacter calcoaceticus NCIB8250, sharing 38%-72% and 58.5%-93.5% respectively. The products encoded by dmp and mop genes convert phenol to catechol. The mph-operon and downstream ORFs, ORF9 and ORF10, sharing high identities to benM and benA, which encode ben-operon regulatory protein and benzoate 1,2-dioxygenase alpha subunit respectively, are separated by ORF8, whose function is unknown. The organization of the mph and ben operons is different from that described previously.     

醋酸钙不动杆菌PHEA-2苯酚降解调控蛋白MphR化学信号域的研究

MphR是醋酸钙不动杆菌PHEA-2调控苯酚代谢的激活蛋白。通过分析苯酚羟化酶启动子的lacZ融合子(PmphK::lacZ)在21种苯酚及苯酚衍生物诱导时的β-半乳糖苷酶活性,确定了调控蛋白MphR化学信号域可以识别苯酚,邻甲酚,间甲酚,邻氯苯酚等酚类衍生物。大肠杆菌中逐段截短MphR化学信号域试验证实,当化学信号域截短后,MphR受苯酚诱导激活转录的功能丧失。结果表明,化学信号域对于苯酚激活转录功能极为重要。     

Benzoate and muconate, structurally dissimilar metabolites, induce expression of catA in Acinetobacter calcoaceticus.

Biosynthetic regulation of catA, the gene encoding catechol 1,2-dioxygenase (EC 1.13.1.1), was studied in an Acinetobacter calcoaceticus mutant strain unable to metabolize benzoate. Benzoate and muconate independently induced the enzyme. In glucose-grown cells, benzoate yielded higher enzyme levels than did muconate, whereas muconate was the more effective inducer in succinate-grown cells.     

Pyrimidine metabolism in Acinetobacter calcoaceticus

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5'-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5'-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5'-monophosphate which is then phosphorylated to uridine-5'-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.     

Concerning the sensitivity of Acinetobacter calcoaceticus

The strains of Acinetobacter calcoaceticus, var. anitratus (A. c. a.) were isolated in the nosocomial environment as an opportune pathogen. The therapy of choice may be determined after in vitro tests. Our results show following therapeutical possibilities: beta-lactam antibiotics--cephalosporins of IIIrd generation (cefotaxime), also combinations of antimicrobials have shown good results: amoxycillin or ticarcillin with clavulanic acid. Best synergistic effect was found in combination ticarcillin-amikacin.     

Expression Analysis of Genes Involved in the RB/E2F Pathway in Astrocytic Tumors

Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression.     

UV-inducible DNA repair in Acinetobacter calcoaceticus

Bacterial mutation frequency after UV irradiation and phage mutation frequency under conditions of W-reactivation were determined in A. calcoaceticus. With the exception of streptomycin resistance, there was no increase in the frequency of the assayed markers above the background level. The increased survival of phage during W-reactivation was not followed by an increase in the frequency of mutation from turbid to clear plaque formers among phage survivors. The findings suggested that the UV-inducible repair pathway in A. calcoaceticus was error free. Post-irradiation incubation of UV-treated culture before phage infection resulted in a further increase of W-reactivation. As chloramphenicol inhibited this response, it was concluded that de novo protein synthesis was involved in the UV-inducible repair pathway in A. calcoaceticus.     

Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader Rhodococcus sp. Strain RHA1

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, exhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1. The gene organization of the RHA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids. Escherichia coli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned benABCD genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1 benABC homologs, suggesting that many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC.     

The surface activity of Acinetobacter calcoaceticus sp. 2CA2

The hydrocarbon metabolizing Acinetobacter calcoaceticus sp. 2CA2 reduces the surface tension of the culture broth during growth on liquid hydrocarbons. This activity, which is not evident during growth on soluble substrates, is associated with the whole cells. Removing the cells from the culture broth increases the surface tension of the liquid phase. The cells when resuspended in water result in a dramatic lowering of the surface tension. Acinetobacter sp. 2CA2 tends to partition between the two liquid phases during growth on hydrocarbons. Both the hydrocarbon bound and nonadhering cells are equally surface active. The whole cells are also able to form and stabilize kerosene-water emulsions. This ability is not related to the lowering of the liquid surface or interfacial tension, since both surface active and nonsurface active cells demonstrated the same emulsifying properties. An extracellular lipopeptide produced during growth on hydrocarbons is not surface active but effectively forms and stabilizes kerosene-water emulsions. The cells and extracellular lipoptide are also effective in de-emulsifying surfactant stabilized test emulsions. The lipopeptide product reduced the half-life of a Tween-Span (TS) stabilized kerosene-water emulsion from 650 to 0.4 h at product concentrations of less than 1% (w/v).     

Purification and Chemical Properties of Acinetobacter calcoaceticus A2 Biodispersan

The extracellular dispersant of Acinetobacter calcoaceticus A2, referred to as biodispersan, was concentrated by ammonium sulfate precipitation and deproteinized by hot phenol treatment. The active component was an anionic polysaccharide (PS-A2). The specific activity of PS-A2 was approximately three times greater than that of crude biodispersan. PS-A2 had a sedimentation constant of 1.39 S, a diffusion coefficient of 18.8 × 10⁻⁸ cm² s⁻¹, and a partial molar volume of 0.65 cm³ g⁻¹, yielding an average molecular weight of 51,400. Titration of the polymer gave two inflection points: pK₁ = 3.1 (1.15 μEq/mg) and pK₂ = 8.0 (0.4 μEq/mg). PS-A2 slowly consumed 1.10 μmol of periodate per mg. The ¹³C nuclear magnetic resonance spectrum of PS-A2 indicated four methyl groups, four carbonyl C atoms, and four signals in the anomeric region (95 to 110 ppm), indicative of the presence of four different monosaccharides. Strong acid hydrolysis of PS-A2 yielded four reducing sugars: glucosamine, a 6-methyl aminohexose, galactosamine uronic acid, and an unidentified amino sugar. Ruthenium red binding to PS-A2 was stoichiometric: 1 molecule of dye bound per 2.0 carboxyl groups.     

Gene transfer in Acinetobacter calcoaceticus NCIB8250

Abstract Filter matings of mutant strains of Acinetobacter calcoaceticus NCIB8250 showed that catabolic and auxotrophic markers were transferred in the absence of a conjugative plasmid. There were no specific 'donor' or 'recipient' strains. Deoxyribonuclease had no effect on the mating system. Some crosses appeared to be highly polarized towards certain parental strains whilst others showed a two-way transfer of genetic markers. There was a high frequency of transfer of the ability to utilize L(+)-mandelate from a mutant of A. calcoaceticus NCIB8250 to certain strains of a second wild-type, EBF65/65, but there was no evidence that the recombinants had acquired another plasmid. This process, whose mechanism is not clear, has some potential in the construction of novel strains but is not likely to be generally useful for mapping purposes and may even prove to be a hazard in the interpretation of results from other genetic techniques.     

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。     

Azide-resistant mutants in Acinetobacter calcoaceticus A2 are defective in protein secretion

Abstract Azide, an inhibitor of ATPase, and a specific inhibitor of protein export was used in order to select for protein secretion mutants in Acinetobacter calcoaceticus A2. Two such mutants were isolated that were azide-resistant and defective in the general protein transport system. The mutation also conferred additional phenotypic changes, including an inability to grow on minimal media or at 40°C. The existence of protein secretion mutants with a selectable phenotype may be useful for the genetic study of protein export.     

Role of cabohydrates in the metabolism of Acinetobacter calcoaceticus

Bacterium N.C.I.B. 8250, a non-saccharolytic strain of Acinetobacter calcoaceticus, is impermeable to extracellular sugars and sugar phosphates but contains intracellularly several glucogenic intermediates.