Cloning of an Erwinia herbicola gene necessary for gluconic acid production and enhanced mineral phosphate solubilization in Escherichia coli HB101: nucleotide sequence and probable involvement in biosynthesis of the coenzyme pyrroloquinoline quinone.
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Escherichia coli is capable of synthesizing the apo-glucose dehydrogenase enzyme (GDH) but not the cofactor pyrroloquinoline quinone (PQQ), which is essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, E. coli does not produce gluconic acid. Evidence is presented to show that the expression of an Erwinia herbicola gene in E. coli HB101(pMCG898) resulted in the production of gluconic acid, which, in turn, implied PQQ biosynthesis. Transposon mutagenesis showed that the essential gene or locus was within a 1.8-kb region of a 4.5-kb insert of the plasmid pMCG898. This 1.8-kb region contained only one apparent open reading frame. In this paper, we present the nucleotide sequence of this open reading frame, a 1,134-bp DNA fragment coding for a protein with an M(r) of 42,160. The deduced sequence of this protein had a high degree of homology with that of gene III (M(r), 43,600) of a PQQ synthase gene complex from Acinetobacter calcoaceticus previously identified by Goosen et al. (J. Bacteriol. 171:447-455, 1989). In minicell analysis, pMCG898 encoded a protein with an M(r) of 41,000. These data indicate that E. coli HB101(pMCG898) produced the GDH-PQQ holoenzyme, which, in turn, catalyzed the oxidation of glucose to gluconic acid in the periplasmic space. As a result of the gluconic acid production, E. coli HB101(pMCG898) showed an enhanced mineral phosphate-solubilizing phenotype due to acid dissolution of the hydroxyapatite substrate. (+info)
Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex.
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The Acinetobacter calcoaceticus-Acinetobacter baumannii complex consists of four genotypically distinct but phenotypically very similar bacterial species or DNA groups: A. calcoaceticus (DNA group 1), A. baumannii (DNA group 2), unnamed DNA group 3 (P. J. M. Bouvet and P. A. D. Grimont, Int. J. Syst. Bacteriol. 36:228-240, 1986), and unnamed DNA group 13 (I. Tjernberg and J. Ursing, APMIS 97:595-605, 1989). Because strains in this complex cause nosocomial outbreaks, it is important to be able to identify them as completely as possible. Ribotyping could provide such identification. Therefore, ribotyping was done on 70 strains in the A. calcoaceticus-A. baumannii complex with known DNA group affiliations by use of restriction enzymes EcoRI, ClaI, and SalI. A nonradioactive digoxigenin-11-dUTP-labeled Escherichia coli rRNA-derived probe was used. With any of the three restriction enzymes, banding patterns that were specific for each DNA group were seen. All 70 strains showed banding patterns that could identify them to the correct DNA group by use of any two of the three enzymes. In addition, banding patterns that could separate strains within any one DNA group were present. The discriminatory index of P. Hunter and M. Gaston (J. Clin. Microbiol. 26:2465-2466, 1988), applied to all strains with the combined results obtained with all three enzymes, revealed a value of 0.99. For strains in each DNA group, the value varied from 0.93 to 0.98. These results indicate the high discriminatory power of the system when used for epidemiological typing. (+info)
Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.
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Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Wurster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal. (+info)
Characterization of a high-affinity iron transport system in Acinetobacter baumannii.
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Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes. (+info)
Endophthalmitis caused by Acinetobacter calcoaceticus. A profile.
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PURPOSE: To report the clinical and microbiological profile of endophthalmitis caused by Acinetobacter calcoaceticus. METHODS: A retrospective study of case series of Acinetobacter calcoaceticus endophthalmitis. Outcome measures included ability to sterilise the eye, anatomical result (clear media and attached retina) and visual recovery (visual acuity > 6/60). RESULTS: Of the 20 cases studied, 10 were cases of postoperative endophthalmitis, 3 were posttraumatic, 6 were endogenous and one was bleb-related endophthalmitis. Specific features of interest observed were relative chronicity of presentation and absence of any obvious predisposing factor in endogenous endophthalmitis cases. All cases could be sterilised except one, which needed evisceration. Cases with postoperative endophthalmitis had better anatomical outcome (7/10 with attached retina and clear media) and visual outcome (4/10 regained vision > 6/18). Higher smear positivity was seen in vitreous samples (72.2%) compared to aqueous samples (37.5%). Culture positivity was higher from the vitreous cavity compared to aqueous. The organism was sensitive to ciprofloxacin in a high percentage (88.9%) of cases. CONCLUSIONS: Visual recovery in Acinetobacter calcoaceticus endophthalmitis is modest. Ciprofloxacin is the antibiotic of choice. (+info)
Purification and characterization of L-2,4-diaminobutyrate decarboxylase from Acinetobacter calcoaceticus.
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Acinetobacter calcoaceticus ATCC 23055 produces a large amount of 1,3-diaminopropane under normal growth conditions. The enzyme responsible, L-2,4-diaminobutyrate (DABA) decarboxylase (EC 4.1.1.-), was purified to electrophoretic homogeneity from this bacterium. The native enzyme had an M(r) of approximately 108,000, with a pI of 5.0, and was a dimer composed of identical or nearly identical subunits with apparent M(r) 53,000. The enzyme showed hyperbolic kinetics with a Km of 1.59 mM for DABA and 14.6 microM for pyridoxal 5'-phosphate as a coenzyme. The pH optimum was in the range 8.5-8.75, and Ca2+ gave a much higher enzyme activity than Mg2+ as a cationic cofactor. N-gamma-Acetyl-DABA, 2,3-diaminopropionic acid, ornithine and lysine were inert as substrates. The enzyme was different in subunit structure, N-terminal amino acid sequence and immunoreactivity from the DABA decarboxylase of Vibrio alginolyticus previously described. (+info)
Microbial hydroxylation of indole to 7-hydroxyindole by Acinetobacter calcoaceticus strain 4-1-5.
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A screening study yielded Acinetobacter calcoaceticus strain 4-1-5, which is capable of hydroxylating indole to 7-hydroxyindole. Strain 4-1-5 grew on terephthalate as the sole source of carbon and energy and hydroxylated indole to 7-hydroxyindole by cometabolism of indole using terephthalate as cosubstrate. Strain 4-1-5 produced 0.574 mM of 7-hydroxyindole at 2.38 mM indole in 24 h with the cell growth. (+info)
Typing and characterization of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex in a Chinese hospital.
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This study was designed to investigate the prevalence of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex (Acb complex) and to type carbapenemases. The relatedness of 45 isolates of carbapenem-resistant Acb complex collected from a clinical setting was analysed by PFGE. The carbapenemases produced by these isolates were typed by IEF, a three-dimensional test, 2-mercaptopropanoic acid inhibition assay, PCR and DNA cloning and sequencing. Results showed that all 45 isolates were resistant to multiple antibiotics including meropenem. The resistance rates to cefoperazone/sulbactam and ampicillin/sulbactam were 2.2 and 6.5%, respectively. About 71.7-78.3% of these isolates were intermediately resistant to cefepime, ceftazidime and cefotaxime. Forty-five isolates were classified into type A (98%) and B (2%) based on their PFGE patterns. Most of type A isolates were from the ICU. Type A was the dominant isolate, including subtypes A1 (22%), A2 (71%), A3 (2%) and A4 (2%). Only one isolate, from the haematology department, belonged to type B. Forty-three isolates (96%) were positive for carbapenemase. One isolate had two bands by IEF, the pIs of which were 6.64 and 7.17. The band with the pI of 6.64 was OXA-23. The other 42 isolates produced two bands with pIs of 6.40 and 7.01 which could not be inhibited by clavulanic acid, cloxacillin or 2-mercaptopropanoic acid. It can be concluded that the prevalent carbapenem-resistant Acb complex isolates from this hospital all had similar beta-lactamase patterns. (+info)