Proteomic analysis of Acinetobacter lwoffii K24 by 2-D gel electrophoresis and electrospray ionization quadrupole-time of flight mass spectrometry

The MS/MS analysis by Electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) was applied to identify proteins in proteome analysis of bacteria whose genomes are not known. The protein identification by ESI-Q-TOF MS was performed sequentially by database search and then de novo sequencing using MS/MS spectra. Soil bacteria having unanalyzed genome, Acinetobacter lwoffii K24 is an aniline degrading bacterium. In this report, we present the results of a comparison between the proteome profile of A. lwoffii K24 cultured in aniline- or succinate-containing media. Protein analysis was performed using two-dimensional gel electrophoresis (2-DE) with pH 3-10 immobilized pH gradient (IPG) strips followed by ESI-Q-TOF MS. More than 780 protein spots were detected by 2-DE from the soluble proteome. Forty-eight of these proteins were expressed exclusively in aniline cultured bacteria, and 81 proteins increased and 162 proteins decreased in aniline-cultured versus succinate cultured A. lwoffii K24. Internal amino acid sequences of 43 major protein spots were successfully determined by ESI-Q-TOF MS to try to identify the bacterial proteins responding to aniline culture condition. Since the A. lwoffii K24 genome is not yet sequenced, many proteins were found to be hypothetical. Comparative proteome analysis of the insoluble protein fractions showed that one novel protein that was strongly induced by succinate-cultured A. lwoffii K24 was repressed under aniline culture conditions. These results suggest that comprehensive analysis of bacterial proteomes by 2-DE and amino acid sequence analysis by ESI-Q-TOF MS is useful for understanding induced novel proteins of biodegrading bacteria.     

Enhanced detection and characterization of protocatechuate 3,4-dioxygenase in Acinetobacter lwoffii K24 by proteomics using a column separation

Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of beta-ketoadipate pathway.     

一株洛菲不动杆菌对碳青霉烯类抗生素耐药机制的研究

目的研究洛菲不动杆菌对亚胺培南、美洛培南耐药的分子机制。方法K-B纸片琼脂扩散法检测洛菲不动杆菌B69对头孢他啶、头孢曲松、环丙沙星、阿米卡星的耐药性,琼脂对倍稀释法检测B69对亚胺培南、美洛培南的最低抑菌浓度;PCR扩增OXA、IMP、VIM型碳青霉烯酶基因,测序确定耐药基因型别;粗提酶水解亚胺培南纸片试验检测酶活性。结果洛菲不动杆菌B69具有多重耐药性;PCR扩增IMP基因阳性,经测序为IMP-8;粗提酶水解亚胺培南。结论产IMP-8型金属β-内酰胺酶是洛菲不动杆菌B69对碳青霉烯耐药的重要机制。     

Gene cloning,sequencing, and expression of an esterase from Acinetobacter lwoffii I6C-1

The esterase-encoding gene, estA, was cloned from Acinetobacter lwoffii I6C-1 genomic DNA into Escherichia coli BL21(DE3) with plasmid vector pET-22b (pEM1). pEM1 has a 4.4-kb EcoRI insert that contained the complete estA gene. A 2.4-kb AvaI- SphI DNA fragment was subcloned (pEM3) and sequenced. estA gene encodes a protein of 366 amino acids (40,687 Da) with a pI of 9.17. The EstA signal peptide was 31 amino acids long, and the mature esterase sequence is 335 amino acids long (37.5 kDa). The conserved catalytic serine residue of EstA is in position 210. The EstA sequence was similar to that of the carboxylesterase from Acinetobacter calcoaceticus (75% identity, 85% similarity), Archaeoglobus fulgidus (37% identity, 59% similarity), and Mycobacterium tuberculosis (35% identity, 51% similarity). These enzymes contained the conserved motif G-X(1)-S-X(2)-G carrying the active-site serine of hydrolytic enzyme. The EstA activity in A. lwoffii I6C-1 remains constant throughout the stationary phase, and the activity in E. coil BL21 (DE3) with pEM1 was similar to A. lwoffii I6C-1.     

Morphological and Biochemical Differentiation of Achromobacter and Moraxella (Debord''s Tribe Mimeae)

To determine the most useful laboratory tests for the differentiation of Achromobacter anitratus, Achromobacter lwoffii, and Moraxella duplex (DeBord's tribe Mimeae), 157 strains of these bacteria, isolated from clinical specimens, were examined for their morphological and biochemical characteristics. There were several differences between these nonfermentative, gram-negative diplococci: Moraxella was nonglucolytic in either infusion base or synthetic base, oxidase-positive, and sensitive to penicillin, whereas Achromobacter produced variable carbohydrate activity, and was oxidase-negative and resistant to penicillin. A. anitratus was distinguished from A. lwoffii in that the former utilized infusion media containing either glucose or 10% lactose, whereas the latter did not. Both species utilized the same carbohydrates in a chemically defined medium, although the latter acted more sluggishly.     

Adhesins of Acinetobacter strains

One of the important factors contributing to the pathogenicity of bacteria is the presence of adhesins on cell surface, which facilitate colonisation in the macroorganism. The presence and type of adhesins occurring in four species of the genus Acinetobacter: A. baumannii (184), A. junii (59), A. lwoffii (65) and A. haemolyticus (22) was determined by haemagglutination test with a 3% suspension of fresh, tannic acid-treated of guinea pig, cow and human group O and AB erythrocytes, with or without the addition of one of sugar inhibitors (D-mannose, alpha-methylmannopyranoside, D-galactose-N-acetyl-D-glucosamine, L-fucose and D-ribose). In strains from all species, adhesines of the mannose-resistant (MR) type dominated. The mannose-sensitive (MS) type was present solely on the surface of one A. lwoffii strains. A. baumannii (36), A. junii (8), A. lwoffii (11) and A. haemolyticus (4) exhibited mannose-resistant hemagglutination in relation to fresh erythrocytes and that reaction was restrained by D-galactose, D-galactose and L-fucose (no other inhibitor used restrained it). The results achieved prove that cell adhesines other than those of MR type must be present on the cell surface. Additional adhesines occurred mainly in strains isolated from the respiratory and urinary tract infection simples, but were not found in isolates from blood cultures.     

Isolation, characterization, and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in the construction of Escherichia coli shuttle plasmids.

Three cryptic plasmids have been discovered in Acinetobacter calcoaceticus BD413. These three plasmids, designated pWM10 (7.4 kb), pWM11 (2.4 kb), and pWM12 (2.2 kb), exhibited extensive homology to one another, as shown by Southern blot hybridization and restriction site analysis data, and also hybridized with three plasmids having slightly different sizes detected in a second strain, A. calcoaceticus BD4. Plasmid pWM11 and a fragment of pWM10 were each subcloned into pUC19, yielding plasmids pWM4 and pWM6, respectively, and were used in a series of inter- and intraspecies transformation experiments. Both plasmids replicated as high-copy-number plasmids in A. calcoaceticus BD413, as well as in strains of Escherichia coli. However, when transformed into the oil-degrading strain Acinetobacter lwoffii RAG-1, both plasmids were maintained at low copy numbers. No modification of the plasmids was detected after repeated transfers between hosts. An analysis of a series of deletions demonstrated that (i) a 185-bp fragment of pWM11 was sufficient to permit replication of the shuttle plasmid in A. calcoaceticus BD413, (ii) the efficiency of transformation of A. calcoaceticus BD413 decreased according to the size of the deletion in the insert by up to 4 orders of magnitude, and (iii) the entire insert was required for transformation and replication in A. lwoffii RAG-1. The sequence of pWM11 contained several small (150- to 300-bp) open reading frames, none of which exhibited any homology to known DNA or protein sequences. In addition, a number of inverted and direct repeats, as well as six copies of the consensus sequence AAAAAAATA previously described for a cryptic plasmid from A. lwoffii (M. Hunger, R. Schmucker, V. Kishan, and W. Hillen, Gene 87:45-51, 1990), were detected. Cloning and expression of the alcohol dehydrogenase regulon from A. lwoffii RAG-1 were accomplished by using the Acinetobacter shuttle plasmid.     

The efficacy of the inorganic copper-based biocide CuWB50 is compromised by hard water

Aims:  We sought to explain the unexpected failure of the inorganic copper-based biocide CuWB50 to effectively decontaminate microfibre cleaning cloths that became contaminated with Acinetobacter lwoffii .
Methods and Results:  CuWB50 was diluted using distilled water or tap water obtained from two different ICUs. Microtitre plate assays were used to determine the minimum inhibitory concentration (MIC) for the implicated A. lwoffii . pH and oxidation-reduction potential (ORP) tests were performed and representative water samples were chemically analysed. When diluted in distilled water, the CuWB50 MIC for A. lwoffii was 9 mg l⁻¹ but in tap water from each ICU it was 37 and 75 mg l⁻¹ at hardness levels of 246 and 296 mg CaCO₃ l⁻¹ respectively. CuWB50-distilled water solutions consistently had a lower pH and higher ORP than CuWB50-tap water solutions.
Conclusions:  Hard water adversely affects the biocidal efficacy of CuWB50.
Significance and Impact of the Study:  Unintentional environmental contamination is a risk when using wet microfibre cloths. This occurred when cloths were stored in CuWB50 overnight combined with the unintentional but erroneous use of tap water. This study emphasizes the need for clearly documented cleaning protocols embedded within a culture of adequate training and constant supervision of cleaning staff.     

A Novel Plasmid pALWVS1.4 from Acinetobacter lwoffii Strain VS15, Carrying the Chloramphenicol Resistance Gene

Microbiology - Sequencing and analysis of the genome of the chloramphenicol-resistant strain Acinetobacter lwoffii VS15, isolated from permafrost, revealed a circular plasmid 11 964 bp in...     

Siderophores and hemolysins in iron aquisition by Acinetobacter genus

In 60 strains of Acinetobacter genus isolated from clinical material belonging to species A. baumannii, A. haemolyticus, A. junii and A. lwoffii a hydroxamate and phenol-catechol class siderophores was identified by chemical and biological testes. A correlation between siderophores production and growth intensity, species affiliation and origin of strains was found.     

Degradation of substituted mandelic acids by meta fission reactions.

A strain of Acinetobacter lwoffii degraded 4-hydroxymandelic and 4-hydroxy-3-methoxymandelic acids to their corresponding benzoates, which were then hydroxylated by specific monooxygenases to yield, respectively, protocatechuic and 3-O-methylgallic acids; these were substrates for meta fission dioxygenases. The product formed from 3-O-methylgallate underwent slow spontaneous cyclization at pH 7 to release methanol.     

Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

Two novel type I catechol 1,2-dioxygenases inducible on aniline media were isolated from Acinetobacter lwoffii K24. Although the two purified enzymes, CD I1 and CD I2, had similar intradiol cleavage activities, they showed different substrate specificities for catechol analogs, physicochemical properties, and amino acid sequences. Two catA genes, catA1 and catA2, encoding by CD I1 and CD I2, respectively, were isolated from the A. lwoffii K24 genomic library by using colony hybridization and PCR. Two DNA fragments containing the catA1 and catA2 genes were located on separate regions of the chromosome. They contained open reading frames encoding 33.4- and 30.4-kDa proteins. The amino acid sequences of the two proteins matched well with previously determined sequences. Interestingly, further analysis of the two DNA fragments revealed the locations of the catB and catC genes as well. Moreover, the DNA fragment containing catA1 had a cluster of genes in the order catB1-catC1-catA1 while the catB2-catA2-catC2 arrangement was found in the catA2 DNA fragment. These results may provide an explanation of the different substrate specificities and physicochemical properties of CD I1 and CD I2.     

宫颈癌患者阴道分泌物非厌氧菌菌群分布及常见菌药敏分析

目的：探讨宫颈癌患者阴道分泌物非厌氧菌菌群分布和常见菌的药敏情况。方法：对87例住院患者的阴道分泌物进行常规培养(不包括厌氧菌、淋病奈瑟菌及支原体培养)和常见菌的药敏测定。结果：87分标本中细菌培养阳性者共86分．阳性率98．8％;共检出菌株161株,革兰阴性菌125株(77．6％),其中嗜麦芽窄食单胞菌和鲁氏不动杆菌分别有49株(39．2％)和43株(34．4％);革兰阳性菌36株(22．4％)．以葡萄球菌和肠球菌为主,分别有18株(50．0％)和9株(25．0％)。药敏结果显示,常见革兰阴性杆菌对氨苄西林的敏感性最差,对亚胺培南、头孢三代的敏感性差别较大．革兰阳性球菌对青霉素的耐药率最高,万古霉素的敏感性最高。结论：宫颈癌患者的阴道分泌物需氧菌群以革兰阴性菌为主,尤以嗜麦芽窄食单胞菌和鲁氏不动杆菌最为常见,临床上根据阴道分泌物菌群分布和药敏情况,在术前采用合适的冲洗液可有效地减少术后感染的发生。     

Design and application of an oligonucleotide microarray for the investigation of compost microbial communities

A microarray consisting of oligonucleotide probes targeting variable regions of the 16S rRNA gene was designed and tested for the investigation of microbial communities in compost. Probes were designed for microorganisms that have been previously reported in the composting process and for plant, animal and human pathogens. The oligonucleotide probes were between 17 and 25 bp in length and included mostly species-specific sequences. Validation of probe specificity and optimization of hybridization conditions were conducted using fluorescently labeled 16S rRNA gene PCR products of pure culture strains. A labeling method employing a Cy3 or Cy5-labeled forward primer together with a phosphate-conjugated reverse primer for the production of single stranded DNA after a digestion step was optimised and used to label target DNA. A combination of two different DNA extraction methods using both physical and chemical lysis was found to give the best DNA yields. Increased hybridization signal intensities were obtained for probes modified with a 12 mer T-spacer. The microarray was found to have a detection limit of 10(3) cells, although in compost spiking experiments, the detection limit was reduced to 10(5) cells. The application of the microarray to compost samples indicated the presence of Streptococcus, Acinetobacter lwoffii, and Clostridium tetani in various compost samples. The presence of A. lwoffii in those compost samples was confirmed by PCR using primers specific for the organism. The aim of this study was to develop a molecular tool that would allow screening for the presence or absence of different microorganisms within compost samples.     

Characteristics of clinical<Emphasis Type="Italic">Acinetobacter</Emphasis> spp. strains

Resistance to 13 antimicrobial agents, resistance to the bactericidal activity of human serum, hydrophobic properties, lipolytic activity and production of histamine were determined in a total of 50 clinicalAcinetobacter spp. strains (A. baumannii, A. lwoffii, A. calcoaceticus, A. haemolyticus). None of the tested isolates showed resistance to meropenem and none ofA. lwoffii, A. calcoaceticus andA. haemolyticus strains were resistant to amikacin. Forty-six strains (92 %) manifested resistance to ampicillin, 90 % to cefuroxime, 68 % to ciprofloxacin, 58 % to piperacillin, gentamicin and cotrimaxazole, 50 % to cefotaxime, 44 % to amikacin, 42 % to ceftazidime, 38 % to piperacillin/tazobactam, 24 % to netilmicin and 16 % to ampicillin/sulbactam. In particular,A. baumannii andA. calcoaceticus strains showed considerable antibiotic resistance. Thirty-one isolates (62 %) showed serum resistance; intermediate sensitivity was found in 19 isolates (38 %). The majority of the strains (72 %) demonstrated a strongly hydrophobic character; 16 % of isolates exhibited moderate hydrophobic properties. All strains showed lipolytic activity; production of histamine was detected in 14 of 43 strains examined.     

7alpha- and 12alpha-Hydroxysteroid dehydrogenases from Acinetobacter calcoaceticus lwoffii: a new integrated chemo-enzymatic route to ursodeoxycholic acid

We report the very efficient biotransformation of cholic acid to 7-keto- and 7,12-diketocholic acids with Acinetobacter calcoaceticus lwoffii. The enzymes responsible of the biotransformation (i.e. 7alpha- and 12alpha-hydroxysteroid dehydrogenases) are partially purified and employed in a new chemo-enzymatic synthesis of ursodeoxycholic acid starting from cholic acid. The first step is the 12alpha-HSDH-mediated total oxidation of sodium cholate followed by the Wolf-Kishner reduction of the carbonyl group to chenodeoxycholic acid. This acid is then quantitatively oxidized with 7alpha-HSDH to 7-ketochenodeoxycholic acid, that was chemically reduced to ursodeoxycholic acid (70% overall yield).     

Acinetobacter calcoaceticus strain with a wide spectrum of utilizing aromatic compounds and carrying a plasmid for resorcin degradation

A bacterial strain was isolated from soil and identified as Acinetobacter calcoaceticus var. lwoffii. The strain can utilize a wide spectrum of aromatic compounds. It carries a transmissive plasmid pBSW13 which determines resorcin utilization via the ortho pathway including the following steps: resorcin-hydroxyhydroquinone-maleylacetate-beta-ketoadipi c acid. The plasmid has been transferred by conjugation into the recipient strains of A. calcoaceticus 5734 CCM rifr, Escherichia coli J-53 met-pro-rifr and Klebsiella sp. Plasmid DNA with a molecular mass close to that of phage gamma was detected by electrophoresis in the donor and recombinant strains. The degradation of other substrates is not a phenotypic expression of the genes of this plasmid.     

低温污水中耐冷微生物的筛选及多样性分析

采用4种分离培养基对新疆乌鲁木齐市污水处理厂低温污水中的耐冷微生物进行分离筛选, 共获得各种耐冷微生物154株, 其中丝状真菌12株, 酵母46株, 放线菌6株, 细菌90株。并对部分菌株的耐受性和产酶特性进行了研究, 获得了耐4%NaCl的菌株44株, 耐0.2%苯酚的16株, 耐0.5% SDS的33株, 具有淀粉酶活性的菌株40株, 蛋白酶活性的30株, 脂酶活性的27株。对其中60株细菌进行16S rRNA基因测序, 序列比对分析表明, 其分属13个属, 其中菌株39与不动杆菌属中的标准菌株Aci